Mammalian cell culture processes for protein production

ABSTRACT

The present invention describes methods and processes for the production of proteins, particularly glycoproteins, by animal cell or mammalian cell culture, preferably, but not limited to, fed-batch cell cultures. In one aspect, the methods comprise the addition of glucocorticoid compound during the culturing period. The addition of glucocorticoid compound sustain a high viability of the cultured cells, and can yield an increased end titer of protein product, and a high quality of protein product, as determined, e.g., by sialic acid content of the produced protein.

This application is a continuation of U.S. application Ser. No.12/897,857, filed Oct. 5, 2010, now allowed, which claims priority toprovisional U.S. Application Ser. No. 61/278,343, filed Oct. 6, 2009,now abandoned.

FIELD OF THE INVENTION

The present invention relates to new processes for culturing mammaliancells which produce a protein product, preferably a glycosylated proteinproduct. Performance of the cell culturing processes result in high cellviability and can also result in high product quality and productivity,extension of the growth phase and reduction of death rate in the deathphase.

BACKGROUND OF THE INVENTION

Animal cell culture, notably mammalian cell culture, is preferably usedfor the expression of recombinantly produced, glycosylated proteins fortherapeutic and/or prophylactic applications. Glycosylation patterns ofrecombinant glycoproteins are important, because the oligosaccharideside chains of glycoproteins affect protein function, as well as theintramolecular interactions between different regions of a protein. Suchintramolecular interactions are involved in protein conformation andtertiary structure of the glycoprotein. (See, e.g., A. Wittwer et al.,1990, Biochemistry, 29:4175-4180; Hart, 1992, Curr. Op. Cell Biol.,4:1017-1023; Goochee et al., 1991, Bio/Technol., 9:1347-1355; and R. B.Parekh, 1991, Curr. Op. Struct. 1:750-754). In addition,oligosaccharides may function to target a particular polypeptide tocertain structures based on specific cellular carbohydrate receptors.(M. P. Bevilacqua et al., 1993, J. Clin. Invest., 91:379-387; R. M.Nelson et al., 1993, J. Clin. Invest., 91:1157-1166; K. E. Norgard etal., 1993, Proc. Natl. Acad. Sci. USA, 90:1068-1072; and Y. Imai et al.,1993, Nature, 361-555-557).

The terminal sialic acid component of a glycoprotein oligosaccharideside chain is known to have an effect on numerous aspects and propertiesof a glycoprotein, including absorption, solubility, thermal stability,serum half life, clearance from the serum, as well as its physical andchemical structure/behavior and its immunogenicity. (A. Varki, 1993,Glycobiology, 3:97-100; R. B. Parekh, Id., Goochee et al., Id., J.Paulson et al., 1989, TIBS, 14:272-276; and A. Kobata, 1992, Eur. J.Biochem., 209:483-501; E. Q. Lawson et al., 1983, Arch. Biochem.Biophys., 220:572-575; and E. Tsuda et al., 1990, Eur. J. Biochem.,188:405-411).

The amount of sialic acid in glycoproteins is affected by two oppositeprocesses: the intracellular additions of sialic acid bysialyltransferase activity and the extracellular removal of sialic acidby sialidase cleavage.

Intracellular addition of sialic acid is the last stage of theglycosylation process that takes place in the trans-Golgi. This involvesthe enzymatic transfer of sialic acid from the nucleotide sugarprecursor, CMP-sialic acid to an available galactose on the emergingglycan structure that is attached to the newly synthesized protein.Possible limitations to the process that might cause incompletesialylation include the availability of CMP-sialic acid, the activity ofthe sialyltransferase enzyme, the amount of the galactose on theemerging glycan structure and the activity of the galactosyltransferaseenzyme. Significant amount of research has been focusing on maximizingsialylation through gene over-expression and enzyme activity enhancementof sialyltransferase and glycosyltransferase. Zhang et al. (BiochimBiophys Acta 1425(3):1998, 441-52) reported that expression of humanα2,6-sialyltransferase in CHO cells with tissue plasminogen activator(tPA) production enhances the α2,6-sialylation of tPA. Weikert et al(Nat Biotechnol 17(11):1999, 1116-21) reported that coexpression ofα2,3-sialyltransferase and β1,4-galactosyltransferase results in greaterthan 90% sialylation of TNK-tPA and TNFR-IgG. Moreover, supplementationwith the proper amount of manganese (Mn²⁺), a cofactor forβ1,4-galactosyltransferase, greatly reduced the amount of rHuEPO in thelower sialylated fraction, increased carbohydrate site occupancy andnarrowed carbohydrate branching (Zhang et al. 1998) to bi-antennarystructures in these lower sialylated species (Crowell et al. BiotechnolBioeng 96(3):538-49, 2007).

The amount of sialic acid in glycoproteins is also affected by theextracellular removal of sialic acid by sialidase cleavage. Gramer andGoochee (Biotechnol Prog 9(4):366-73, 1993) have demonstrated anincrease of lactate dehydrogenase (LDH), which signified an increase inthe cell lysis, correlated with an increase of extracellular sialidaseactivity in CHO perfusion cultures. Gu et al (Biotechnol Bioeng55(2):390-8, 1997) also illustrate a remarkable loss of terminal sialicacids of interferon-γ (IFN-γ) along with decrease in CHO cell viabilityand concomitant increase of dead cells throughout long-term batchcultivation.

Consequently, It is essential to delay the onset of cell death andimprove cell viability to reduce or avoid this degradation effect

In general, protein expression levels in mammalian cell culture-basedsystems are considerably lower than in microbial expression systems, forexample, bacterial or yeast expression systems. However, bacterial andyeast cells are limited in their ability to optimally express highmolecular weight protein products, to properly fold a protein having acomplex steric structure, and/or to provide the necessarypost-translational modifications to mature an expressed glycoprotein,thereby affecting the immunogenicity and clearance rate of the product.

As a consequence of the limitations of the culturing of animal ormammalian cells, particularly animal or mammalian cells which producerecombinant products, the manipulation of a variety of parameters hasbeen investigated, including the employment of large-scale culturevessels; altering basic culture conditions, such as incubationtemperature, dissolved oxygen concentration, pH, and the like; the useof different types of media and additives to the media; and increasingthe density of the cultured cells. In addition, process development formammalian cell culture would benefit from advances in the ability toextend run times to increase final product concentration whilemaintaining high product quality. An important product quality parameteris the degree and completeness of the glycosylation structure of apolypeptide product, with sialic acid content commonly used as a measureof glycoprotein quality.

Run times of cell culture processes, particularly non-continuousprocesses, are usually limited by the remaining viability of the cells,which typically declines over the course of the run. The maximumpossible extension of high cell viabilities is therefore desired.Product quality concerns also offer a motivation for minimizingdecreases in viable cell density and maintaining high cell viability, ascell death can release sialidases to the culture supernatant, which mayreduce the sialic acid content of the protein expressed. Proteinpurification concerns offer yet another motivation for minimizingdecreases in viable cell density and maintaining high cell viability.The presence of cell debris and the contents of dead cells in theculture can negatively impact on the ability to isolate and/or purifythe protein product at the end of the culturing run. By keeping cellsviable for a longer period of time in culture, there is thus aconcomitant reduction in the contamination of the culture medium bycellular proteins and enzymes, e.g., cellular proteases and sialidasesthat can cause degradation and ultimate reduction in quality of thedesired glycoprotein produced by the cells.

Various parameters have been investigated to achieve high cell viabilityin cell cultures. One parameter involved a single lowering of theculture temperature following initial culturing at 37° C. (for example,Roessler et al., 1996, Enzyme and Microbial Technology, 18:423-427; U.S.Pat. Nos. 5,705,364 and 5,721,121 to T. Etcheverry et al., 1998; U.S.Pat. No. 5,976,833 to K. Furukawa et al., 1999; U.S. Pat. No. 5,851,800to L. Adamson et al.; WO 99/61650 and WO 00/65070 to Genentech, Inc.; WO00/36092 to Biogen, Inc.; and U.S. Pat. No. 4,357,422 to Girard et al.).

Other parameters investigated involved the addition of components to theculture. The growth factor inhibitor suramin was shown to preventapoptosis during exponential growth of CHO K1:CycE cells (Zhangi et al.,Biotechnol. Prog. 2000, 16, 319-325). However, suramin did not protectagainst apoptosis during the death phase. As a result, suramin wascapable of maintaining high viability during the growth phase, but didnot allow for an extension of culture longevity. The same authors reportthat for the CHO 111-10PF cell line, dextran sulfate and polyvinylsulfate could, similarly to suramin, increase day 3 viable cell densityand viability relative to the control culture. The effect of dextransulfate or polyvinyl sulfate during the death phase was however notreported. Suramin, dextran sulfate and polyvinyl sulfate were alsoreported to be effective at preventing cell aggregation.

The effects of supplementing insect cell culture media withdexamethasone or N-acetylmannosarnine on complex glycosylation ofproteins, including the addition of terminal sialic acid residues toN-linked oligosaccharides, prepared via baculovirus expression vectorsystem (BEVS) is disclosed in U.S. Pat. No. 6,472,175 to Boyce ThompsonInstitute For Plant Research, Inc. (Ithaca, N.Y.), 2002.

Protein therapeutics are inherently heterogeneous owing to their size,complexity of structure, and the nature of biological production(Chirino and Mire-Sluis, Nat Biotechnol. 2004; 22:1383-1391). Even inthe “pure” protein solution, there will be some percentage of lowmolecular weight fragments, high molecular weight species, and variousdegrees of chemical modifications. The formation of high molecularweight species is usually due to protein aggregation, which is a commonissue encountered during manufacture of biologics. Typically, thepresence of aggregates is considered to be undesirable because of theconcern that the aggregates may lead to an immunogenic reaction or maycause adverse events on administration (Cromwell et al, AAPS J. 2006;8:E572-579). Although some types of aggregates of biologics may functionnormally, it is still important to maintain consistency in productquality since product consistency is a prerequisite for regulatoryapproval.

Aggregates of proteins may arise from several mechanisms and occur ateach stage during the manufacturing process. In cell culture, secretedproteins may be exposed to the conditions that are unfavorable forprotein stability; but more often, accumulation of high amounts ofprotein may lead to intracellular aggregation owing to either theinteractions of unfolded protein molecules or to inefficient recognitionof the nascent peptide chain by molecular chaperones responsible forproper folding (Cromwell et al, AAPS J. 2006; 8:E572-579). In theendoplasmic reticulum (ER) of cells, disulfide bond of newly synthesizedprotein is formed in an oxidative environment. Under normal condition,protein sulfhydryls are reversibly oxidized to protein disulfides andsulfenic acids, but the more highly oxidized states such as the sulfinicand sulfonic acid forms of protein cysteines are irreversible (Thomasand Mallis, Exp Gerontol. 2001; 36:1519-1526). Hyper-oxidized proteinsmay contain incorrect disulfide bonds or have mixed disulfide bonds withother luminal ER proteins; in either case it leads to protein improperfolding and aggregation. It is therefore crucial to maintain a properlycontrolled oxidative environment in the ER. In this regard, Cuozzo andKaiser (Nat Cell Biol. 1999; 1:130-135) initially demonstrated that inyeasts glutathione buffered against ER hyperoxidation and later onChakravarthi and Bulleid (J Biol Chem. 2004; 279:39872-39879) confirmedthat in mammalian cells glutathione was also required to regulate theformation of native disulfide bonds within proteins entering thesecretory pathway.

With increasing product concentration in the culture, it can be observedin cell culture processes that the product quality decreases, asdetermined by the measured sialic acid content of the oligosaccharideglycostructure. Usually, a lower limit for an acceptable sialic acidcontent exists as determined by drug clearance studies. High abundanceof a protein produced by cells in culture is optimally accompanied byhigh quality of the protein that is ultimately recovered for an intendeduse.

Recombinantly produced protein products that are properly glycosylatedare increasingly becoming medically and clinically important for use astherapeutics, treatments and prophylactics. Therefore, the developmentof reliable cell culture processes that economically and efficientlyachieve an increased final protein product concentration, in conjunctionwith a high level of product quality, such as is determined by sialicacid content, fulfills both a desired and needed goal in the art.

SUMMARY OF THE INVENTION

The present invention provides new processes for the production ofproteins, preferably recombinant protein products, more preferablyglycoprotein products, by animal or mammalian cell cultures. These newprocesses achieve increased viable cell density at the late phase, cellviability, productivity and sialic acid content and decreased proteinaggregation.

One aspect of this invention concerns the addition of glucocorticoid tothe media. In this aspect, cell culture processes of this invention canadvantageously achieve an enhanced specific productivity, e.g.,glycoprotein, as well as an enhanced sialic acid content of theglycoprotein produced by the cultured cells. More specifically, inaccordance with this invention, addition of glucocorticoid during thecell culturing period sustains a high cell viability of the cells in theculture and can provide a high quantity and quality of produced productthroughout an entire culture run. Also, according to one aspect of theinvention, addition of glucocorticoid to the culturing processes canadvantageously allow for an extension of the production phase of theculture. During the extended production phase, the titer of the desiredproduct is increased; the product quality, as characterized by sialicacid content, is maintained at a high level; protein aggregation levelis maintained at lower level and cell viability is also maintained at ahigh level. In addition, the extended production phase associated withthe culturing processes of the invention allows for the production ofproduct beyond that which is produced during a standard productionphase.

In one particular aspect, the present invention provides a process (ormethod) in which the specific productivity is enhanced, the proteinaggregation level was reduced and the sialic acid content of theproduced glycoprotein is higher, by the addition of glucocorticoid.Glucocorticoid compound preferably is dexamethasone. In accordance withthis particular aspect, the addition of glucocorticoid sustains a highcell viability of the culture, thereby enabling an extended productionphase during which the titer of product, preferably recombinant product,is increased and the product quality, as characterized by sialic acidcontent, is maintained at high level. The addition of glucocorticoid canminimize the prevailing trade-off between protein titer and sialic acidcontent in the production of product during the cell culture process.Thus, the addition of glucocorticoid provides a positive effect onenhancing an important performance parameter of the culturing process,i.e., the mathematical product of “end (i.e., final) titer”×“end (i.e.,final) sialic acid”×“monomer content” (“end titer×end sialic acid”×“endmonomer content).

In one aspect of this invention, glucocorticoid compound is added to aculture at the time of inoculation or at a time after inoculation thatis before the beginning of the initial death phase, or is during theinitial growth phase, or is during the second half of the initial growthphase, or is on or about the end of the initial growth phase. Inaccordance with this aspect of the invention, the growth phase isextended and/or the onset of the death phase is delayed for a period oftime, such as several days.

In another preferred aspect of this invention and as further describedherein, the newly developed cell culture processes involving theaddition of a glucocorticoid compound, are especially suitable for theproduction of soluble CTLA4 molecules and soluble CTLA4 mutant molecule,such as CTLA4lg and L104EA29Ylg, by host cells genetically engineered toexpress and produce these proteins. Preferred embodiments of the presentinvention encompass the culturing of cells producing CTLA4lg andL104EA29Ylg involving the addition of a glucocorticoid compound duringthe culturing run to achieve large amounts of high quality CTLA4lg andL104EA29Ylg products, as determined by sialic acid measurement and/orlow protein aggregation of the final products.

Further aspects, features and advantages of the present invention willbe appreciated upon a reading of the detailed description of theinvention and a consideration of the drawings/figures.

DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the expressions of β1,4-galactosyltransferase (1A) andα2,3-sialyltransferase (1B) generally increase with dexamethasone (DEX)concentration in the DEX treated CHO cells as described in Example 1.DEX was added on the 2^(nd) day after inoculation to the cultures atconcentrations from 0.1 to 10 10 μM. On the fifth day after inoculation,cell samples were collected and whole cell lysates were prepared,separated on a 4-15% gradient gel and probed with the antibody for eachglycotransferase. The blot was then reprobed with the beta-actinantibody to assess equal loading.

FIG. 2 shows the cell protective effect of DEX resulting in reducedsialidase activity in the culture supernatant as described in Example 1.Cell viability (2A) and absorbance of supernatant sialidase activityassay (2B) profiles along the culture period between DEX treated anduntreated cultures. DEX treatments at concentrations of 1 μM wereinitiated on day 2. Values reflect the mean and standard deviation ofdata from five experiments.

FIG. 3 shows improvements in glycoprotein sialylation in the culturestreated with glucocorticoid analogs, hydrocortisone (HYC) andprednisolone (PRD) as described in Example 1. Normalized total sialicacid content (3A) and normalized N-linked sialylated species fraction(3B) of cultures treated with DEX, HYC and PRD, respectively. Treatmentwas initiated on the second day after inoculation in a concentration of0.1, 1 and 10 μM in the medium for each compound. Values of eachparameter are reported as average±difference/2 (n=2)

FIG. 4 shows that the enhancement of sialylation by DEX was blocked byglucocorticoid antagonist RU-486 as described in Example 1. Normalizedtotal sialic acid content (4A) and normalized N-linked sialylatedspecies fraction (4B) in the presence and absence of RU-486. RU-486, at0 or 1 μM, was introduced into cell culture suspension 48 hours afterinoculation. 0.1, 1 and 10 μM of DEX was then added into the cultures 24hours later. Values of each parameter are reported asaverage±difference/2 (n=2).

FIG. 5 shows that fed-batch cultures of DEX treated CHO cells in 5-Lbioreactors resulted in increased sialic acid content and sialylatedspecies fractions as described in Example 1. Normalized total sialicacid content (5A) and normalized N-linked sialylated species fraction(5B) of untreated and treated cultures throughout the cultivations.Values of each parameter are reported as average±standard deviation(n=3). The normalized value is the actual value divided by an arbitraryvalue.

FIG. 6 shows the capability of DEX to improve glycoprotein sialylationat 7, 10, and 20-L scale bioreactors as described in Example 1.Normalized final total sialic acid molar ratio versus normalizedsialylated fraction of DEX treated and untreated cultures from differentruns are summarized. The normalized value is the actual value divided byan arbitrary value.

FIG. 7 shows the reduced percentage of high molecular weight (HMW)species in the IgG-fusion proteins produced by CHO cells treated withdexamethasone (DEX) as described in Example 2. 7A, all cells wereinitially cultured together in the same shake flask for two days andthen divided into two groups, with half of them receiving a single doseof DEX at the final concentration of 1 μM in basal medium. Thesupernatants were collected on Day 10 and purified before SEC-HPLCanalysis to determine percentages of HMW species. Data points are means(±S.D.) of the results from five shake flasks in a single experiment.**P<0.01 as compared to the control (CON). 7B, DEX at variousconcentrations was added to the CHO cell cultures on day 2 andsupernatants were collected on day 10. Data points are means of theresults obtained from duplicate flasks. 7C, all the cultures wereinitiated at the same time, but 1 μM DEX was added on different day,giving different incubation time as indicated, when the cells wereharvested at the same time on day 10. Data points are means of theresults from duplicate flasks.

FIG. 8 shows the increased expression of glutathione reductase in theCHO cells treated with dexamethasone (DEX) as described in Example 2.DEX at various concentrations was added to the cell cultures on theinoculation day and cell samples were collected on day 5. Whole celllysates were separated on a 4-15% gradient gel. After detection ofglutathione reductase, the same blot was used to detect β-actin forsample loading comparison.

FIG. 9 shows the reduced percentage of HMW species in the IgG-fusionproteins incubated with GSH in vitro as described in Example 2. Reducedglutathione (GSH) at 0, 1 and 3 mM final concentrations was added toTris-acetate buffered (pH 7.5) solution of purified IgG-fusion proteins,and the mixture of GSH and proteins was incubated at 37° C. for 1 hbefore SEC-HPLC analysis. Data points are means (±S.D.) of fourdeterminations from two experiments. *P<0.05 as compared to the control(0 mM GSH).

FIG. 10 shows the attenuated effects of dexamethasone (DEX) in thepresence of glucocorticoid receptor antagonist RU-486 as described inExample 2. 10A, whole cell lysates were prepared from untreated HepG-2and CHO cells and separated on a 4-15% gradient gel. HepG-2 sample wasused as human origin control for primary antibody validation purpose.10B, RU-486 at 1 μM was introduced into the culture one day after theinoculation (day 1) and RU-486-pretreated CHO cells were divided on day2, with half of them receiving a single dose of DEX at the finalconcentration of 0.1 μM in basal medium. Cell samples were collected onday 10; all the other procedures were the same as those in FIG. 2. 10C,RU-486 at 1 μM was introduced into the cultures on day 1 and DEX wasthen added to RU-486-pretreated cultures at the final concentration ofeither 0.1 μM or 1 μM on day 2. Supernatants were collected on day 10.Data points are means of the results from duplicate flasks.

FIG. 11 shows that cell death is inhibited by DEX in cultures of CHOcells in serum free medium as described in Example 3. Dose-responsecurves of the effect of DEX on viable cell density (11A) and viability(11B) with treatments initiated on day 2. Time-course curves of theeffect of DEX on viable cell density (11C) and viability (11D) with 1 μMtreatment concentration. Each value is the mean of data obtained fromthe experiments done in duplicate.

FIG. 12 shows CHO cell specific growth rate is reduced while cellspecific productivity is increased by DEX as described in Example 3. Theeffects of DEX on CHO cell specific growth rate (12A), normalizedvolumetric productivity (12B) and normalized cell specific productivity(12B). DEX treatments were initiated on day 2. Values reflect the meanand standard deviation of data from five experiments. The normalizedvalue is the actual value divided by an arbitrary value.

FIG. 13 shows the upregulation of anti-apoptotic gene GILZ in theDEX-treated CHO cells was confirmed by qRT-PCR and western blotanalysis, as described in Example 3. (13A) DEX was added into thetriplicate cultures on the 2^(nd) day after inoculation in the finalconcentration of 0 and 1 μM, respectively. mRNA samples was extracted on5^(th) and 8^(th) after inoculation. Values of each parameter arereported as average±standard deviation (n=3). (13B) DEX was added intothe triplicate cultures on the 2^(nd) day after inoculation in the finalconcentration of 0, and 1 μM, respectively. Cell samples were collectedand whole cell lysates were prepared on 5^(th) and 8^(th) afterinoculation.

FIG. 14 shows the death-suppression action of dexamethasone involvesGILZ and glucocorticoid receptor, as described in Example 3 The percentincrease (compared with no DEX treatment) of final viability (14A), thefold change of GILZ gene expression (14B) and GILZ protein expressioninduced by DEX (14C) in the presence and absence of RU-486. RU-486, at 0or 1 μM, was introduced into cell culture suspension 48 hours afterinoculation, 0, 0.1 and 1 μM of DEX was then added into the cultures 24hours later. Cells were collected for viability, qRT-PCR and westernblotting analysis. Each value reported in panel A is the mean of dataobtained from the duplicate experiments. Each value reported in panel Bis the mean and standard deviation of data obtained from the triplicateexperiments.

FIG. 15 shows fed-batch cultures of DEX treated CHO cells in 10-Lbioreactors result in an improved VCD, viability, titer and sialic acidmolar ratio as described in Example 3. Viable cell density (15A),viability (15B) and normalized titer (15C) profiles of untreated cultureand treated cultures with DEX treatment initiated on day 2 and day 7.The normalized value is the actual value divided by an arbitrary value.

FIG. 16 shows the effects of DEX on the cell growth of CHO cell culturewith CTLA4lg secretion. Dose-response curves of the effect of DEX onviable cell density (16A) and viability (16B) treated with DEX in thefinal concentration of 0, 0.001, 0.01, 0.1, 1 and 10 μM, respectively.Treatment was initiated on the second day after inoculation and eachvalue is the mean of data obtained from the experiments done induplicate.

FIG. 17 shows the effects of DEX on sialic acid molar ratio and HMWlevel of CTLA4lg. The figure shows final total sialic acid molar ratio(17A) and HMW species (17B) of cultures treated with DEX in the finalconcentration of 0, 0.001, 0.01, 0.1, 1 and 10 μM, respectively.Treatment was initiated on the second day after inoculation and eachvalue is the mean of data obtained from the experiments done induplicate. Values reported in panel A are normalized values, which arethe actual value divided by an arbitrary value.

FIG. 18 shows the feasibility of including DEX in the large scalerecombinant glycoprotein production, as described in Example 5. Thefigure shows the viable cell density (18A), viability (18B), normalizedtiter (18C) and normalized sialic acid content (18D) of the recombinantglycoprotein produced at 7-L (n=16) and 500-L (n=6) and 5000-L (n=3)scales, respectively. Values reflect the mean and standard deviation ofdata from multiple experiments at each scale. The normalized value isthe actual value divided by an arbitrary value. The same divisor wasutilized for normalization in all scales.

FIG. 19 depicts a nucleotide sequence (SEQ ID NO:1) and encoded aminoacid sequence (SEQ ID NO:2) of a CTLA4lg having a signal peptide, a wildtype amino acid sequence of the extracellular domain of CTLA4 startingat methionine at position +1 to aspartic acid at position +124, orstarting at alanine at position −1 to aspartic acid at position +124,and an Ig region.

FIG. 20 depicts a nucleotide sequence (SEQ ID NO:3) and encoded aminoacid sequence (SEQ ID NO:4) of a CTLA4 mutant molecule (L104EA29Ylg)having a signal peptide, a mutated extracellular domain of CTLA4starting at methionine at position +1 and ending at aspartic acid atposition +124, or starting at alanine at position −1 and ending ataspartic acid at position +124, and an Ig region.

FIG. 21 depicts the nucleic acid sequence (SEQ ID NO:5) and encodedcomplete amino acid sequence (SEQ ID NO:6) of human CTLA4 receptor(referred to as “wild type” CTLA4 herein) fused to the oncostatin Msignal peptide (position −26 to −2). (U.S. Pat. Nos. 5,434,131 and5,844,095).

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes new processes for the production ofproteins, preferably recombinant protein products, more preferablyglycoprotein products, in mammalian or animal cell culture. Theseprocesses achieve increased viable cell density, cell viability,productivity and sialic acid content and decreased protein aggregation.

In one embodiment, the invention is directed to a cell culturing processcomprising: culturing host cells which express a protein of interest;and adding glucocorticoid compound to the cell culture.

Glucocorticoid compounds include, but are not limited to, hydrocortisone(available from Sigma-Aldrich, St. Louis, Mo.), prednisone (availablefrom Sigma-Aldrich), prednisolone (available from Sigma-Aldrich),methylprednisolone (available from Sigma-Aldrich), dexamethasone(available from Sigma-Aldrich), betamethasone (available fromSigma-Aldrich), triamcinolone (available from Sigma-Aldrich),fludrocortisone acetate (available from Sigma-Aldrich). The compoundsare readily available from the listed sources, or readily obtainablethrough means known to one of skill in the art.

Preferred glucocorticoid compounds include but not limited tohydrocortisone, prednisolone, betamethasone and dexamethasone. Mostpreferred is dexamethasone.

In one embodiment of the invention, glucocorticoid compound is added atinoculation or may be a component of the basal medium. Inoculation takesplace on day 0.

In one embodiment of the invention, glucocorticoid compound is added ata time after inoculation, i.e. it is not present in the basal medium andnot present at inoculation. Preferably, the glucocorticoid compound isadded on day 1 of the culture or later

In accordance with the invention, glucocorticoid compound may be addedto the cell culture one time, two times, three times, or any number oftimes during the specified time period. One or more glucocorticoidcompounds may be used in conjunction. That is, any given single additionof a glucocorticoid compound may include the addition of one or moreother glucocorticoid compounds. Similarly, if there is more than oneaddition of a glucocorticoid compound, different glucocorticoidcompounds may be added at the different additions. Additional compoundsand substances, including glucocorticoid compounds, may be added to theculture before, with or after the addition of glucocorticoidcompound—either during or not during the specified time period. In apreferred embodiment, there is a single, i.e. one time, addition ofglucocorticoid compound. In a preferred embodiment, one glucocorticoidcompound is added.

In accordance with the invention, glucocorticoid compound may be addedto the cell culture by any means. Means of adding glucocorticoidcompound include, but are not limited to, dissolved in DMSO, dissolvedin organic solvent, dissolved in water, dissolved in culture medium,dissolved in feed medium, dissolved in a suitable medium, in the form inwhich it is obtained or any combination thereof.

Preferably, DEX is added as a solution where the DEX is dissolved inethanol that is then diluted with water for further use (i.e. such asadding DEX to the feed medium).

In accordance with the invention, glucocorticoid compound is added tobring the concentration in the culture to an appropriate level. Asnonlimiting examples, glucocorticoid compound is added to aconcentration of 1 nM-1 mM. Preferably glucocorticoid compound is addedto a concentration of 1 nM-0.1 μM or 0.1 μM-10 μM, more preferably about5 nM-15 nM or 0.5 μM-5 μM, more preferably about 10 nM or 1 μM targetamounts.

In accordance with the invention, the culture may be run for any lengthof time after addition of glucocorticoid compound. The culture run timemay be determined by one of skill in the art, based on relevant factorssuch as the quantity and quality of recoverable protein, and the levelof contaminating cellular species (e.g. proteins and DNA) in thesupernatant resulting from cell lysis, which will complicate recovery ofthe protein of interest.

In particular embodiments of the cell culturing process and method ofincreasing cell viability of the invention, glucocorticoid compound isadded at a time after inoculation that is before the beginning of theinitial death phase. Preferably, glucocorticoid compound is added at atime after inoculation that is during the initial growth phase. Morepreferably, glucocorticoid compound is added during the second half theinitial growth phase. More preferably, glucocorticoid compound is addedon or about the end of the initial growth phase.

The initial growth phase refers to the growth phase that is observed inthe absence of the specified addition of glucocorticoid compound. Theinitial death phase refers to the death phase that is observed in theabsence of the specified addition of glucocorticoid compound.

The initial growth phase may end when the initial death phase begins, orthere may be a stationary phase of any length between the initial growthphase and the initial death phase.

For example, in a cell culture in which the initial growth phase is fromday 0 to day 6 and the initial death phase begins on day 7, in aparticular embodiment glucocorticoid compound is added at a time afterinoculation and before day 7. In a specific embodiment, glucocorticoidcompound is added after inoculation and by day 6. In a specificembodiment, glucocorticoid compound is added between days 1 and 6. Inanother specific embodiment, glucocorticoid compound is added with thefeed medium on days 3-6. In other specific embodiments, glucocorticoidcompound is added on about day 2, or on day 2.

It has been found (see Example 3) that when carrying the presentinvention the viability of the cell culture is prolonged. A condition,such as addition of glucocorticoid compound, causes prolonged cellviability if cell viability in the culture is higher for a period oftime in the presence of the condition than in the absence of thecondition.

Thus, in other embodiments, the invention is directed to (1) a cellculturing process, and (2) a method of prolonging cell viability in aculture comprising: culturing host cells which express a protein ofinterest; and adding glucocorticoid compound to the cell culture;wherein the cell viability of the cell culture is prolonged.

It has been found (see Example 3), that when glucocorticoid compound isadded at a time after inoculation and before the beginning of theinitial death phase, the death rate of the death phase may be reduced,less than that of the death phase observed in the absence of theaddition of glucocorticoid compound.

Thus, in other embodiments, the invention is directed to (1) a cellculturing process, and (2) a process for reducing the death rate of thedeath phase of a cell culture comprising: culturing host cells whichexpress a protein of interest; and adding glucocorticoid compound to thecell culture at a time after inoculation that is before the beginning ofthe initial death phase; wherein the death rate of the death phase isreduced. In more particular embodiments, the invention is directed to(1) a cell culturing process, and (2) a process for reducing the deathrate of the death phase of a cell culture comprising: culturing hostcells which express a protein of interest; and adding glucocorticoidcompound to the cell culture at a time after inoculation that is duringthe initial growth phase; wherein the death rate of the death phase isdelayed. In more particular embodiments the invention is directed to (1)a cell culturing process, and (2) a process for reducing the death rateof the death phase of a cell culture comprising: culturing host cellswhich express a protein of interest; and adding glucocorticoid compoundto the cell culture during the second half of the initial growth phase;wherein the death rate of the death phase is reduced. In otherparticular embodiments the invention is directed to a process forreducing the death rate of the death phase of a cell culture comprising:culturing host cells which express a protein of interest; and addingglucocorticoid compound to the cell culture on or about the end of theinitial growth phase; wherein the death rate of the death phase isdelayed.

Example 3 also demonstrates that hydrocortisone (HYC), prednisolone(PRD) and dexamethasone (DEX) all show a dose-dependent cell protectiveeffect in treated cell cultures when compared with untreated cellcultures. However, higher concentrations of HYC and PRD was required toachieve the same level of cell protective effect, which is consistentwith their potency differences (i.e. HYC and PRD are only 5% and 20% aspotent as DEX)

Run times of cell culture processes, particularly non-continuousprocesses, are usually limited by the remaining viable cell density,which decreases during the death phase. Longer run times may allowhigher product titers to be achieved. Product quality concerns alsooffer a motivation for reducing death rate, as cell death can releasesialidases to the culture supernatant, which may reduce the sialic acidcontent of the protein expressed. Protein purification concerns offeryet another motivation for delaying or arresting the death phase. Thepresence of cell debris and the contents of dead cells in the culturecan negatively impact on the ability to isolate and/or purify theprotein product at the end of the culturing run.

It has been found (see Example 2), that addition of glucocorticoidcompound to the cell culture reduces the aggregation of the proteins ofinterest.

Thus, in other embodiments, the invention is directed to (1) a cellculturing process, and (2) a process for reducing the percentage ofprotein aggregation comprising: culturing host cells which express aprotein of interest; and adding glucocorticoid compound to the cellculture; wherein the percentage of high molecular weight species isdecreased.

It has been found (see Example 1), that addition of glucocorticoidcompound to the cell culture improves sialylation of the proteins ofinterest by enhancing total sialic acid content and increasingpercentage of sialylated species.

Example 1 also demonstrates that hydrocortisone (HYC), prednisolone(PRD) and dexamethasone (DEX) all show a dose-dependent sialylationimprovement in treated cell cultures when compared with untreated cellcultures. However, a higher concentration of HYC and PRD was required toachieve the same level of improvement, which is consistent with theirpotency differences.

Thus, in other embodiments, the invention is directed to (1) a cellculturing process, and (2) a process for increasing the percentage ofsialylated species comprising: culturing host cells which express aprotein of interest; and adding glucocorticoid compound to the cellculture; wherein the percentage of sialylated species is increased.

Thus, in other embodiments, the invention is directed to (1) a cellculturing process, and (2) a process for increasing total sialic acidcontent comprising: culturing host cells which express a glycoprotein ofinterest; and adding glucocorticoid compound to the cell culture;wherein the total sialic acid content is increased.

Thus, in other embodiments, the invention is directed to (1) a cellculturing process, and (2) a process for reducing de-sialylation rate ofglycoproteins in cell culture comprising: culturing host cells whichexpress a glycoprotein of interest; and adding glucocorticoid compoundto the cell culture; wherein the de-sialylation rate is decreased.

Techniques and Procedures Relating to Glycoprotein Purification andAnalysis

In the culturing methods encompassed by the present invention, theprotein produced by the cells is typically collected, recovered,isolated, and/or purified, or substantially purified, as desired, at theend of the total cell culture period using isolation and purificationmethods as known and practiced in the art. Preferably, protein that issecreted from the cultured cells is isolated from the culture medium orsupernatant; however, protein can also be recovered from the host cells,e.g., cell lysates, using methods that are known and practiced in theart, and as further described below.

The complex carbohydrate comprising the glycoprotein produced by theprocesses of this invention can be routinely analyzed, if desired, byconventional techniques of carbohydrate analysis. For example,techniques such as lectin blotting, well-known in the art, revealproportions of terminal mannose, or other sugars such as galactose.Termination of mono-, bi-, tri-, or tetra-antennary oligosaccharide bysialic acids can be confirmed by release of sugars from the proteinusing anhydrous hydrazine or enzymatic methods and fractionation ofoligosaccharides by ion-exchange chromatography, size exclusionchromatography, or other methods that are well-known in the art.

The pl of the glycoprotein can also be measured, before and aftertreatment with neuraminidase, to remove sialic acids. An increase in plfollowing neuraminidase treatment indicates the presence of sialic acidson the glycoprotein. Carbohydrate structures typically occur on theexpressed protein as N-linked or O-linked carbohydrates. The N-linkedand O-linked carbohydrates differ primarily in their core structures.N-linked glycosylation refers to the attachment of the carbohydratemoiety via GlcNAc to an asparagine residue in the peptide chain. TheN-linked carbohydrates all contain a commonMan1-6(Man1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-R core structure, where R in thiscore structure represents an asparagine residue. The peptide sequence ofthe protein produced will contain an asparagine-X-serine,asparagine-X-threonine, and asparagine-X-cysteine, wherein X is anyamino acid except proline.

In contrast, O-linked carbohydrates are characterized by a common corestructure, which is GalNAc attached to the hydroxyl group of a threonineor serine. Of the N-linked and O-linked carbohydrates, the mostimportant are the complex N- and O-linked carbohydrates. Such complexcarbohydrates contain several antennary structures. The mono-, bi-,tri-, and tetra-, outer structures are important for the addition ofterminal sialic acids. Such outer chain structures provide foradditional sites for the specific sugars and linkages that comprise thecarbohydrates of the protein products.

The resulting carbohydrates can be analyzed by any method known in theart. Several methods are known in the art for glycosylation analysis andare useful in the context of the present invention. These methodsprovide information regarding the identity and the composition of theoligosaccharide attached to the produced peptide. Methods forcarbohydrate analysis useful in connection with the present inventioninclude, but are not limited to, lectin chromatography; HPAEC-PAD, whichuses high pH anion exchange chromatography to separate oligosaccharidesbased on charge; NMR; Mass spectrometry; HPLC, GPO; monosaccharidecompositional analysis; and sequential enzymatic digestion.

In addition, methods for releasing oligosaccharides are known andpracticed in the art. These methods include 1) enzymatic methods, whichare commonly performed using peptide-N-glycosidaseF/endo-β-galactosidase; 2) β elimination methods, using a harsh alkalineenvironment to release mainly O-linked structures; and 3) chemicalmethods using anhydrous hydrazine to release both N- and O-linkedoligosaccharides. Analysis can be performed using the followingsteps: 1. Dialysis of the sample against deionized water to remove allbuffer salts, followed by lyophilization. 2. Release of intactoligosaccharide chains with anhydrous hydrazine. 3. Treatment of theintact oligosaccharide chains with anhydrous methanolic HCl to liberateindividual monosaccharides as O-methyl derivatives. 4. N-acetylation ofany primary amino groups. 5. Derivatization to yieldper-O-trimethylsilyl methyl glycosides. 6. Separation of thesederivatives by capillary gas-liquid chromatography (GLC) on a CP-SIL8column. 7. Identification of individual glycoside derivatives byretention time from the GLC and mass spectroscopy, compared to knownstandards. 8. Quantification of individual derivatives by FID with aninternal standard (13-O-methyl-D-glucose).

Neutral and amino sugars can be determined by high performanceanion-exchange chromatography combined with pulsed amperometricdetection (HPAE-PAD Carbohydrate System; Dionex Corp.). For instance,sugars can be released by hydrolysis in 20% (v/v) trifluoroacetic acidat 100° C. for 6 hours. Hydrolysates are then dried by lyophilization orwith a Speed-Vac (Savant Instruments). Residues are then dissolved in 1%sodium acetate trihydrate solution and analyzed on an HPLC-AS6 column(as described by Anumula et al., 1991, Anal. Biochem., 195:269-280).

Alternatively, immunoblot carbohydrate analysis can be performed. Inthis procedure protein-bound carbohydrates are detected using acommercial glycan detection system (Boehringer), which is based on theoxidative immunoblot procedure described by Haselbeck et al. (1993,Glycoconjugate J., 7:63). The staining protocol recommended by themanufacturer is followed except that the protein is transferred to apolyvinylidene difluoride membrane instead of a nitrocellulose membraneand the blocking buffers contain 5% bovine serum albumin in 10 mM Trisbuffer, pH 7.4, with 0.9% sodium chloride. Detection is carried out withanti-digoxigenin antibodies linked with an alkaline phosphate conjugate(Boehringer), 1:1000 dilution in Tris buffered saline using thephosphatase substrates, 4-nitroblue tetrazolium chloride, 0.03% (w/v)and 5-bromo-4 chloro-3-indoyl-phosphate 0.03% (w/v) in 100 mM Trisbuffer, pH 9.5, containing 100 mM sodium chloride and 50 mM magnesiumchloride. The protein bands containing carbohydrate are usuallyvisualized in about 10 to 15 minutes.

Carbohydrate associated with protein can also be analyzed by digestionwith peptide-N-glycosidase F. According to this procedure the residue issuspended in 14 μL of a buffer containing 0.18% SDS, 18 mMbeta-mercaptoethanol, 90 mM phosphate, 3.6 mM EDTA, at pH 8.6, andheated at 100° C. for 3 minutes. After cooling to room temperature, thesample is divided into two equal parts. One part, which is not treatedfurther, serves as a control. The other part is adjusted to about 1%NP-40 detergent followed by the addition of 0.2 units ofpeptide-N-glycosidase F (Boehringer). Both samples are warmed at 37° C.for 2 hours and then analyzed by SDS-polyacrylamide gel electrophoresis.

In addition, the sialic acid content of the glycoprotein product isassessed by conventional methods. For example, sialic acid can beseparately determined by a direct colorimetric method (Yao et al., 1989,Anal. Biochem., 179:332-335), preferably using triplicate samples.Another method of sialic acid determination involves the use ofthiobarbaturic acid (TBA), as described by Warren et al. (1959, J. Biol.Chem., 234:1971-1975). Yet another method involves high performancechromatography, such as described by H. K. Ogawa et al. (1993, J.Chromatography, 612:145-149).

Illustratively, for glycoprotein recovery, isolation and/orpurification, the cell culture medium or cell lysate is centrifuged toremove particulate cells and cell debris. The desired polypeptideproduct is isolated or purified away from contaminating soluble proteinsand polypeptides by suitable purification techniques. The followingprocedures provide exemplary, yet nonlimiting purification methods forproteins: separation or fractionation on immunoaffinity or ion-exchangecolumns; ethanol precipitation; reverse phase HPLC; chromatography on aresin, such as silica, or cation exchange resin, e.g., DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, e.g., Sephadex G-75, Sepharose; protein A sepharosechromatography for removal of immunoglobulin contaminants; and the like.Other additives, such as protease inhibitors (e.g., PMSF or proteinaseK) can be used to inhibit proteolytic degradation during purification.It will be understood by the skilled practitioner that purificationmethods for a given polypeptide of interest may require modificationswhich allow for changes in the polypeptide expressed recombinantly incell culture. Those purification procedures that can select forcarbohydrates and enrich for sialic acid are particularly preferred,e.g., ion-exchange soft gel chromatography, or HPLC using cation- oranion-exchange resins, in which the more acidic fraction(s) is/arecollected.

Cells, Proteins and Cell Cultures

In the cell culture processes or methods of this invention, the cellscan be maintained in a variety of cell culture media. i.e., basalculture media, as conventionally known in the art. For example, themethods are applicable for use with large volumes of cells maintained incell culture medium, which can be supplemented with nutrients and thelike. Typically, “cell culturing medium” (also called “culture medium”)is a term that is understood by the practitioner in the art and is knownto refer to a nutrient solution in which cells, preferably animal ormammalian cells, are grown and which generally provides at least one ormore components from the following: an energy source (usually in theform of a carbohydrate such as glucose); all essential amino acids, andgenerally the twenty basic amino acids, plus cysteine; vitamins and/orother organic compounds typically required at low concentrations; lipidsor free fatty acids, e.g., linoleic acid; and trace elements, e.g.,inorganic compounds or naturally occurring elements that are typicallyrequired at very low concentrations, usually in the micromolar range.Cell culture medium can also be supplemented to contain a variety ofoptional components, such as hormones and other growth factors, e.g.,insulin, transferrin, epidermal growth factor, serum, and the like;salts, e.g., calcium, magnesium and phosphate, and buffers, e.g., HEPES;nucleosides and bases, e.g., adenosine, thymidine, hypoxanthine; andprotein and tissue hydrolysates, e.g., hydrolyzed animal protein(peptone or peptone mixtures, which can be obtained from animalbyproducts, purified gelatin or plant material); antibiotics, e.g.,gentamycin; and cell protective agents, e.g., a Pluronic polyol(Pluronic F68). Preferred is a cell nutrition medium that is serum-freeand free of products or ingredients of animal origin.

As is appreciated by the practitioner, animal or mammalian cells arecultured in a medium suitable for the particular cells being culturedand which can be determined by the person of skill in the art withoutundue experimentation. Commercially available media can be utilized andinclude, for example, Minimal Essential Medium (MEM, Sigma, St. Louis,Mo.); Ham's F10 Medium (Sigma); Dulbecco's Modified Eagles Medium (DMEM,Sigma); RPMI-1640 Medium (Sigma); HyClone cell culture medium (HyClone,Logan, Utah); and chemically-defined (CD) media, which are formulatedfor particular cell types, e.g., CD-CHO Medium (Invitrogen, Carlsbad,Calif.). To the foregoing exemplary media can be added theabove-described supplementary components or ingredients, includingoptional components, in appropriate concentrations or amounts, asnecessary or desired, and as would be known and practiced by thosehaving in the art using routine skill.

In addition, cell culture conditions suitable for the methods of thepresent invention are those that are typically employed and known forbatch, fed-batch, or continuous culturing of cells, with attention paidto pH, e.g., about 6.5 to about 7.5; dissolved oxygen (O₂), e.g.,between about 5-90% of air saturation and carbon dioxide (CO₂),agitation and humidity, in addition to temperature. As an illustrative,yet nonlimiting, example, a suitable cell culturing medium for thefed-batch processes of the present invention comprises a modified CD-CHOMedium (Invitrogen, Carlsbad, Calif.). A feeding medium can also beemployed, such as modified eRDF medium (Invitrogen, Carlsbad, Calif.).Preferred is a feeding medium also containing glucocorticoid, e.g.dexamethasone.

Animal cells, mammalian cells, cultured cells, animal or mammalian hostcells, host cells, recombinant cells, recombinant host cells, and thelike, are all terms for the cells that can be cultured according to theprocesses of this invention. Such cells are typically cell linesobtained or derived from mammals and are able to grow and survive whenplaced in either monolayer culture or suspension culture in mediumcontaining appropriate nutrients and/or growth factors. Growth factorsand nutrients that are necessary for the growth and maintenance ofparticular cell cultures are able to be readily determined empiricallyby those having skill in the pertinent art, such as is described, forexample, by Barnes and Sato, (1980, Cell, 22:649); in Mammalian CellCulture, Ed. J. P. Mather, Plenum Press, N Y, 1984; and in U.S. Pat. No.5,721,121.

Numerous types of cells can be cultured according to the methods of thepresent invention. The cells are typically animal or mammalian cellsthat can express and secrete, or that can be molecularly engineered toexpress and secrete, large quantities of a particular protein, moreparticularly, a glycoprotein of interest, into the culture medium. Itwill be understood that the glycoprotein produced by a host cell can beendogenous or homologous to the host cell. Alternatively, andpreferably, the glycoprotein is heterologous, i.e., foreign, to the hostcell, for example, a human glycoprotein produced and secreted by aChinese hamster ovary (CHO) host cell. Also preferably, mammalianglycoproteins, i.e., those originally obtained or derived from amammalian organism, are attained by the methods the present inventionand are preferably secreted by the cells into the culture medium.

Examples of mammalian glycoproteins that can be advantageously producedby the methods of this invention include, without limitation, cytokines,cytokine receptors, growth factors (e.g., EGF, HER-2, FGF-α, FGF-β,TGF-α, TGF-β, PDGF. IGF-1, IGF-2, NGF, NGF-β); growth factor receptors,including fusion or chimeric proteins. Other nonlimiting examplesinclude growth hormones (e.g., human growth hormone, bovine growthhormone); insulin (e.g., insulin A chain and insulin B chain),proinsulin; erythropoietin (EPO); colony stimulating factors (e.g.,G-CSF, GM-CSF, M-CSF); interleukins (e.g., IL-1 through IL-12), vascularendothelial growth factor (VEGF) and its receptor (VEGF-R); interferons(e.g., IFN-α, β, or γ) tumor necrosis factor (e.g., TNF-α and TNF-β) andtheir receptors, TNFR-1 and TNFR-2; thrombopoietin (TPO); thrombin;brain natriuretic peptide (BNP); clotting factors (e.g., Factor VIII,Factor IX, von Willebrands factor, and the like); anti-clotting factors;tissue plasminogen activator (TPA), e.g., urokinase or human urine ortissue type TPA; follicle stimulating hormone (FSH); luteinizing hormone(LH); calcitonin; CD proteins (e.g., CD3, CD4, CD8, CD28, CD19, etc.);CTLA proteins (e.g., CTLA4); T-cell and B-cell receptor proteins; bonemorphogenic proteins (BNPs, e.g., BMP-1, BMP-2, BMP-3, etc.);neurotrophic factors, e.g., bone derived neurotrophic factor (BDNF);neurotrophins, e.g., 3-6; renin; rheumatoid factor; RANTES; albumin;relaxin; macrophage inhibitory protein (e.g., MIP-1, MIP-2); viralproteins or antigens; surface membrane proteins; ion channel proteins;enzymes; regulatory proteins; antibodies; immunomodulatory proteins,(e.g., HLA, MHC, the B7 family); homing receptors; transport proteins;superoxide dismutase (SOD); G-protein coupled receptor proteins (GPCRs);neuromodulatory proteins; Alzheimer's Disease associated proteins andpeptides, (e.g., A-beta), and others as known in the art. Fusionproteins and polypeptides, chimeric proteins and polypeptides, as wellas fragments or portions, or mutants, variants, or analogues of any ofthe aforementioned proteins and polypeptides are also included among thesuitable proteins, polypeptides and peptides that can be produced by themethods of the present invention.

Nonlimiting examples of animal or mammalian host cells suitable forharboring, expressing, and producing proteins for subsequent isolationand/or purification include Chinese hamster ovary cells (CHO), such asCHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec.Genet., 12:555-556; and Kolkekar et al., 1997, Biochemistry,36:10901-10909), CHO-K1 Tet-On cell line (Clontech), CHO designatedECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG,Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1/SF designated ECACC93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductasenegative CHO cells (CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl.Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121);monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCCCRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293 cellssubcloned for growth in suspension culture, Graham et al., 1977, J. Gen.Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkeykidney cells (CV1, ATCC CCL-70); African green monkey kidney cells(VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4,Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells(HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lungcells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mousemammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells(BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci.,383:44-68); MCR 5 cells; FS4 cells. Preferred are CHO cells,particularly, CHO/-DHFR cells.

The cells suitable for culturing in the methods and processes of thepresent invention can contain introduced, e.g., via transformation,transfection, infection, or injection, expression vectors (constructs),such as plasmids and the like, that harbor coding sequences, or portionsthereof, encoding the proteins for expression and production in theculturing process. Such expression vectors contain the necessaryelements for the transcription and translation of the inserted codingsequence. Methods which are well known to and practiced by those skilledin the art can be used to construct expression vectors containingsequences encoding the produced proteins and polypeptides, as well asthe appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in J. Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubelet al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

Control elements, or regulatory sequences, are those non-translatedregions of the vector, e.g., enhancers, promoters, 5′ and 3′untranslated regions, that interact with host cellular proteins to carryout transcription and translation. Such elements can vary in theirstrength and specificity. Depending on the vector system and host cellutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, can be used. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferred. The constructs for use in protein expressionsystems are designed to contain at least one promoter, an enhancersequence (optional, for mammalian expression systems), and othersequences as necessary or required for proper transcription andregulation of gene expression (e.g., transcriptional initiation andtermination sequences, origin of replication sites, polyadenylationsequences, e.g., the Bovine Growth Hormone (BGH) poly A sequence).

As will be appreciated by those skilled in the art, the selection of theappropriate vector, e.g., plasmid, components for proper transcription,expression, and isolation of proteins produced in eukaryotic (e.g.,mammalian) expression systems is known and routinely determined andpracticed by those having skill in the art. The expression of proteinsby the cells cultured in accordance with the methods of this inventioncan placed under the control of promoters such as viral promoters, e.g.,cytomegalovirus (CMV), Rous sarcoma virus (RSV), phosphoglycerol kinase(PGK), thymidine kinase (TK), or the α-actin promoter. Further,regulated promoters confer inducibility by particular compounds ormolecules, e.g., the glucocorticoid response element (GRE) of mousemammary tumor virus (MMTV) is induced by glucocorticoids (V. Chandler etal., 1983, Cell, 33:489-499). Also, tissue-specific promoters orregulatory elements can be used (G. Swift et al., 1984, Cell,38:639-646), if necessary or desired.

Expression constructs can be introduced into cells by a variety of genetransfer methods known to those skilled in the art, for example,conventional gene transfection methods, such as calcium phosphateco-precipitation, liposomal transfection, microinjection,electroporation, and infection or viral transduction. The choice of themethod is within the competence of the skilled practitioner in the art.It will be apparent to those skilled in the art that one or moreconstructs carrying DNA sequences for expression in cells can betransfected into the cells such that expression products aresubsequently produced in and/or obtained from the cells.

In a particular aspect, mammalian expression systems containingappropriate control and regulatory sequences are preferred for use inprotein expressing mammalian cells of the present invention. Commonlyused eukaryotic control sequences for use in mammalian expressionvectors include promoters and control sequences compatible withmammalian cells such as, for example, the cytomegalovirus (CMV) promoter(CDM8 vector) and avian sarcoma virus (ASV), (πLN). Other commonly usedpromoters include the early and late promoters from Simian Virus 40(SV40) (Fiers et al., 1973, Nature, 273:113), or other viral promoterssuch as those derived from polyoma, Adenovirus 2, and bovine papillomavirus. An inducible promoter, such as hMTII (Karin et al., 1982, Nature,299:797-802) can also be used.

Examples of expression vectors suitable for eukaryotic host cellsinclude, but are not limited to, vectors for mammalian host cells (e.g.,BPV-1, pHyg, pRSV, pSV2, pTK2 (Maniatis); pIRES (Clontech); pRc/CMV2,pRc/RSV, pSFV1 (Life Technologies); pVPakc Vectors, pCMV vectors, pSG5vectors (Stratagene), retroviral vectors (e.g., pFB vectors(Stratagene)), pcDNA-3 (Invitrogen), adenoviral vectors;Adeno-associated virus vectors, baculovirus vectors, yeast vectors(e.g., pESC vectors (Stratagene)), or modified forms of any of theforegoing. Vectors can also contain enhancer sequences upstream ordownstream of promoter region sequences for optimizing gene expression.

A selectable marker can also be used in a recombinant vector (e.g., aplasmid) to confer resistance to the cells harboring (preferably, havingstably integrated) the vector to allow their selection in appropriateselection medium. A number of selection systems can be used, includingbut not limited to, the Herpes Simplex Virus thymidine kinase (HSV TK),(Wigler et al., 1977, Cell, 11:223), hypoxanthine-guaninephosphoribosyltransferase (HGPRT), (Szybalska and Szybalski, 1992, Proc.Natl. Acad. Sci. USA, 48:202), and adenine phosphoribosyltransferase(Lowy et al., 1980, Cell, 22:817) genes, which can be employed in tk-,hgprt-, or aprt-cells (APRT), respectively.

Anti-metabolite resistance can also be used as the basis of selectionfor the following nonlimiting examples of marker genes: dhfr, whichconfers resistance to methotrexate (Wigler et al., 1980, Proc. Natl.Acad. Sci. USA, 77:357; and O'Hare et al., 1981, Proc. Natl. Acad. Sci.USA, 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA, 78:2072); neo,which confers resistance to the aminoglycoside G418 (Clinical Pharmacy,12:488-505; Wu and Wu, 1991, Biotherapy, 3:87-95; Tolstoshev, 1993, Ann.Rev. Pharmacol. Toxicol., 32:573-596; Mulligan, 1993, Science,260:926-932; Anderson, 1993, Ann. Rev. Biochem., 62:191-21; May, 1993,TIB TECH, 11(5):155-215; and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene, 30:147). Methods commonly knownin the art of recombinant DNA technology can be routinely applied toselect the desired recombinant cell clones, and such methods aredescribed, for example, in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, N Y (1993); Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY; inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, N Y (1994); Colberre-Garapin et al., 1981.J. Mol. Biol., 150:1, which are incorporated by reference herein intheir entireties.

In addition, the expression levels of an expressed protein molecule canbe increased by vector amplification (for a review, see Bebbington andHentschel, “The use of vectors based on gene amplification for theexpression of cloned genes in mammalian cells in DNA cloning”, Vol. 3,Academic Press, New York, 1987). When a marker in the vector systemexpressing a protein is amplifiable, an increase in the level ofinhibitor present in the host cell culture will increase the number ofcopies of the marker gene. Since the amplified region is associated withthe protein-encoding gene, production of the protein will concomitantlyincrease (Crouse et al., 1983, Mol. Cell. Biol., 3:257).

Vectors which harbor glutamine synthase (GS) or dihydrofolate reductase(DHFR) encoding nucleic acid as the selectable markers can be amplifiedin the presence of the drugs methionine sulphoximine or methotrexate,respectively. An advantage of glutamine synthase based vectors is theavailability of cell lines (e.g., the murine myeloma cell line, NSO)which are glutamine synthase negative. Glutamine synthase expressionsystems can also function in glutamine synthase expressing cells (e.g.,CHO cells) by providing additional inhibitor to prevent the functioningof the endogenous gene.

Vectors that express DHFR as the selectable marker include, but are notlimited to, the pSV2-dhfr plasmid (Subramani et al., Mol. Cell. Biol.1:854 (1981). Vectors that express glutamine synthase as the selectablemarker include, but are not limited to, the pEE6 expression vectordescribed in Stephens and Cockett, 1989, Nucl. Acids. Res., 17:7110. Aglutamine synthase expression system and components thereof are detailedin PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are incorporated by reference herein in theirentireties. In addition, glutamine synthase expression vectors that canbe used in accordance with the present invention are commerciallyavailable from suppliers, including, for example, Lonza Biologics, Inc.(Portsmouth, N.H.).

In a particular embodiment, a nucleic acid sequence encoding a solubleCTLA4 molecule or a soluble CTLA4 mutant molecule can be inserted into avector designed for expressing foreign sequences in a eukaryotic host.The regulatory elements of the vector can vary according to theparticular eukaryotic host. Vectors which express the soluble CTLA4 orsoluble CTLA4 mutant in eukaryotic host cells can include enhancersequences for optimizing protein expression.

Types of Cell Cultures

For the purposes of understanding, yet without limitation, it will beappreciated by the skilled practitioner that cell cultures and culturingruns for protein production can include three general types; namely,continuous culture, batch culture and fed-batch culture. In a continuousculture, for example, fresh culture medium supplement (i.e., feedingmedium) is provided to the cells during the culturing period, while oldculture medium is removed daily and the product is harvested, forexample, daily or continuously. In continuous culture, feeding mediumcan be added daily and can be added continuously, i.e., as a drip orinfusion. For continuous culturing, the cells can remain in culture aslong as is desired, so long as the cells remain alive and theenvironmental and culturing conditions are maintained.

In batch culture, cells are initially cultured in medium and this mediumis neither removed, replaced, nor supplemented, i.e., the cells are not“fed” with new medium, during or before the end of the culturing run.The desired product is harvested at the end of the culturing run.

For fed-batch cultures, the culturing run time is increased bysupplementing the culture medium one or more times daily (orcontinuously) with fresh medium during the run, i.e., the cells are“fed’ with new medium (“feeding medium”) during the culturing period.Fed-batch cultures can include various feeding regimens and times, forexample, daily, every other day, every two days, etc., more than onceper day, or less than once per day, and so on. Further, fed-batchcultures can be fed continuously with feeding medium.

The desired product is then harvested at the end of theculturing/production run. The present invention preferably embracesfed-batch cell cultures in which a glucocorticoid compound is added at atime after inoculation.

According to the present invention, cell culture can be carried out, andglycoproteins can be produced by cells, under conditions for the largeor small scale production of proteins, using culture vessels and/orculture apparatuses that are conventionally employed for animal ormammalian cell culture. As is appreciated by those having skill in theart, tissue culture dishes, T-flasks and spinner flasks are typicallyused on a laboratory scale. For culturing on a larger scale, (e.g., 500L, 5000 L, and the like, for example, as described in commonly-assignedU.S. Pat. No. 7,541,164, U.S. Pat. No. 7,332,303, and U.S. applicationSer. No. 12/086,786, filed Dec. 19, 2006, the contents of which areincorporated by reference herein in their entirety) proceduresincluding, but not limited to, a fluidized bed bioreactor, a hollowfiber bioreactor, roller bottle culture, or stirred tank bioreactorsystems can be used. Microcarriers may or may not be used with theroller bottle or stirred tank bioreactor systems. The systems can beoperated in a batch, continuous, or fed-batch mode. In addition, theculture apparatus or system may or may not be equipped with a cellseparator using filters, gravity, centrifugal force, and the like.

Phases of Cell Culture and Associated Parameters

The term “inoculation” refers to the addition of cells to startingmedium to begin the culture.

The “growth phase” of a culture is the phase during which the viablecell density at any time point is higher than at any previous timepoint.

The “stationary phase” of a culture is the phase during which the viablecell density is approximately constant (i.e. within measuring error)over a time period of any length.

The “death phase” of a culture is the phase that comes after the growthphase or after the growth phase and the stationary phase, and duringwhich the viable cell density at any time point is lower than at anyprevious time point during that phase.

In a “growth-associated” culture process, such as cases where aglucocorticoid compound causes an extended growth phase, the productionphase may start during the extended growth phase.

In a “non-growth” associated culture process, the production phase ofcell culture may be the stationary phase.

Preferably, the culture medium is supplemented (“fed”) during theproduction phase to support continued protein production, particularlyin an extended production phase, and to attain ample quantities of highquality glycoprotein product (as exemplified and/or determined by a highlevel of end sialic acid content upon protein recovery). Feeding canoccur on a daily basis, or according to other schedules to support cellviability and protein production.

The culturing process according to the present invention may result inmore viable cell survival until the end of the culturing period.Accordingly, in some embodiments, the more cells that survive, the morecells that are producing the desired product. This, in turn, results ina greater accumulated amount of the product at the end of the culturingprocess, with the rate of protein production by individual cells, i.e.,cell specific productivity, remaining the same. Cell specificproductivity or cell specific rate, as known in the art, typicallyrefers to the specific expression rate of product produced per cell, orper measure of cell mass or volume. Cell specific productivity ismeasured in grams of protein produced per cell per day, for example, andcan be measured according to an integral method involving the followingformulae:

dP/dt=q _(p) X, or

P=q _(p)∫₀ ^(t) Xdt

where q_(p) is the cell specific productivity constant; X is the numberof cells or cell volume, or cell mass equivalents; and dP/dt is the rateof protein production. Thus, q_(p) can be obtained from a plot ofproduct concentration versus time integral of viable cells (∫₀ ^(t) Xdt“viable cell days”). According to this formula, when the amount ofglycoprotein product produced is plotted against the viable cell days,the slope is equivalent to the cell specific rate. Viable cells can bedetermined by several measures, for example, biomass, 02 uptake rate,lactase dehydrogenase (LDH), packed cell volume or turbidity. (e.g.,U.S. Pat. No. 5,705,364 to T. Etcheverry et al.)

Production of Soluble CTLA4 Molecules and Soluble CTLA4 Mutant Moleculesby the Culturing Methods of the Present Invention

In other embodiments encompassed by the present invention, the cellculture methods of the invention are utilized to produce a soluble CTLA4molecule or a soluble CTLA4 mutant molecule, as described below. Asoluble CTLA4 molecule is preferably a CTLA4 fusion protein, preferablya CTLA4lg. More preferred is CTLA4lg that comprises amino acids −1 to357 or +1 to 357 as shown in FIG. 19. A soluble CTLA4 mutant molecule ispreferably L104EA29Ylg that comprises amino acids −1 to 357 or +1 to 357as shown in FIG. 20. The cell culture methods involving extendedproduction phases for protein product are especially suitable forgenerating high quality and large amounts of soluble CTLA4 molecules andsoluble CTLA4 mutant molecules, by their host cells in culture.

In a preferred embodiment, CTLA4lg is produced by recombinantlyengineered host cells. The CTLA4lg fusion protein can be recombinantlyproduced by CHO cells transfected with a vector containing the DNAsequence encoding CTLA4lg. (See, U.S. Pat. No. 5,844,095 to P. S.Linsley et al). The CTLA4lg fusion protein is produced in high quantityand is appropriately sialylated when cultured in accordance with theprocesses of this invention. The invention affords the production ofhigh levels of recoverable protein product, e.g., sialylated CTLA4lgprotein product. In another preferred embodiment, the soluble CTLA4mutant molecule L104EA29Ylg that comprises amino acids −1 to 357 or +1to 357 as shown in FIG. 20 is produced by the cell culture methods ofthe present invention.

A ligand for CTLA4 is a B7 molecule. As used herein, “ligand” refers toa molecule that specifically recognizes and binds another molecule. Theinteraction of a molecule and its ligand can be regulated by theproducts of the culturing processes of this invention. For example,CTLA4 interaction with its ligand B7 can be blocked by theadministration of CTLA4lg molecules. As other examples, the interactionof Tumor Necrosis Factor (TNF) with its ligand, TNF receptor (TNFR), canbe blocked by administration of etanercept or other TNF/TNFR blockingmolecules.

Wild type CTLA4 or “non-mutated CTLA4” has the amino acid sequence ofnaturally occurring, full length CTLA4 as shown in FIG. 20 (and also asdescribed in U.S. Pat. Nos. 5,434,131, 5,844,095, and 5,851,795,incorporated herein by reference in their entirety), or any portionthereof that recognizes and binds a B7 molecule, or interferes with a B7molecule, so that binding to CD28 and/or CTLA4 (e.g., endogenous CD28and/or CTLA4) is blocked. Wild type CTLA4 comprises particular portions,including, for example, the extracellular domain of wild type CTLA4beginning with methionine at position +1 and ending at aspartic acid atposition +124, or the extracellular domain of wild type CTLA4 beginningwith alanine at position −1 and ending at aspartic acid at position +124as shown in FIG. 21.

The naturally occurring wild type CTLA4 is a cell surface protein havingan N-terminal extracellular domain, a transmembrane domain, and aC-terminal cytoplasmic domain. The extracellular domain binds to atarget molecule, such as a B7 molecule. In a cell, the naturallyoccurring, wild type CTLA4 protein is translated as an immaturepolypeptide, which includes a signal peptide at the amino, orN-terminal, end. The immature polypeptide undergoes post-translationalprocessing, which includes cleavage and removal of the signal peptide togenerate a CTLA4 cleavage product having a newly generated N-terminalend that differs from the N-terminal end in the immature form. Oneskilled in the art will appreciate that additional post-translationalprocessing may occur, which removes one or more of the amino acids fromthe newly generated N-terminal end of the CTLA4 cleavage product. Themature CTLA4 protein may start at methionine at position +1 or alanineat position −1. The mature form of the CTLA4 molecule includes theextracellular domain or any portion thereof, which binds to B7.

A CTLA4 mutant molecule, as used herein, refers to a molecule comprisingwild type CTLA4 as shown in FIG. 21, or any portion or derivativethereof that has a mutation, or multiple mutations, in the wild typeCTLA4 sequence, preferably in the extracellular domain of wild typeCTLA4, and binds B7. A CTLA4 mutant molecule has a sequence that it issimilar, but not identical, to the sequence of wild type CTLA4 molecule,but still binds B7. The mutations can include one or more amino acidresidues substituted with an amino acid having conservative (e.g., aleucine substituted for an isoleucine) or non-conservative (e.g., aglycine substituted with a tryptophan) structure or chemical properties,amino acid deletions, additions, frameshifts, or truncations.

CTLA4 mutant molecules can include a non-CTLA4 molecule therein orattached thereto, i.e., CTLA4 mutant fusion proteins. The mutantmolecules can be soluble (i.e., circulating) or they can be bound to acell surface (membrane-bound). CTLA4 mutant molecules includeL104EA29Ylg and those described in U.S. Application Ser. Nos. 60/214,065and 60/287,576; in WO 01/92337 A2; in U.S. Pat. Nos. 6,090,914,5,844,095, 7,094,874 and 5,773,253; and as described in R. J. Peach etal., 1994, J Exp Med, 180:2049-2058.) CTLA4 mutant molecules can besynthetically or recombinantly produced.

CTLA4lg is a soluble fusion protein comprising an extracellular domainof wild type CTLA4, or a portion thereof that binds B7, joined to animmunoglobulin (Ig) molecule, or a portion thereof. The extracellulardomain of CTLA4 or portion thereof is joined to an Ig moiety comprisingall or a portion of an immunoglobulin molecule, preferably all or aportion of an immunoglobulin constant region such as all or a portion ofIgCγ1 (IgCgamma1), IgCγ2 (IgCgamma2), IgCγ3 (IgCgamma3), IgCγ4(IgCgamma4), IgCμ (IgCmu), IgCα1 (IgCalpha1), IgCα2 (IgCalpha2), IgCδ(IgCdelta) or IgCε (IgCepsilon), rendering the fusion molecule soluble.The Ig moiety can include the hinge, CH2 and CH3 domains, or the CH1,hinge, CH2 and CH3 domains, of the aforementioned constant regions orother constant regions. Preferably, the Ig moiety is human or monkey andcomprises the hinge, CH2 and CH3 domains. Most preferably the Ig moietycomprises the hinge, CH2 and CH3 domains of human IgCγ1, or consists ofthe hinge, CH2 and CH3 domains of human IgCγ1. In an Ig moiety ofCTLA4lg, the Ig constant region or portion thereof can be mutated, thusresulting in a reduction of its effector functions (see, e.g., U.S. Pat.Nos. 5,637,481, 5,844,095 and 5,434,131). As used herein, the terms Igmoiety, Ig constant region, Ig C (constant) domain, IgCγ1 (IgCgamma1),IgCγ2 (IgCgamma2), IgCγ3 (IgCgamma3), IgCγ4 (IgCgamma4), IgCμ (IgCmu),IgCα1 (IgCalpha1), IgCα2 (IgCalpha2), IgCδ (IgCdelta) or IgCε(IgCepsilon), include both native sequences and sequences that have beenmutated, such as, for example, sequences having mutations in theconstant region that reduce effector function.

A particular embodiment related to CTLA4 comprises the extracellulardomain of wild type CTLA4 starting at methionine at position +1 andending at aspartic acid at position +124, or starting at alanine atposition −1 to aspartic acid at position +124; a junction amino acidresidue glutamine at position +125; and an immunoglobulin portionencompassing glutamic acid at position +126 through lysine at position+357, as shown in FIG. 19. DNA encoding this CTLA4lg was deposited onMay 31, 1991, in the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, under the provisions of theBudapest Treaty, and has been accorded ATCC accession number ATCC 68629;P. Linsley et al., 1994, Immunity 1:793-80. A CHO cell line expressingCTLA4lg was deposited on May 31, 1991 in ATCC under identificationnumber CRL-10762. The soluble CTLA4lg molecules produced according tothe methods described herein may or may not include a signal (leader)peptide sequence. FIGS. 19 and 20 include an illustration of a signal(leader) peptide sequence. Typically, the molecules do not include asignal peptide sequence.

L104EA29Ylg is a fusion protein that is a soluble CTLA4 mutant moleculecomprising an extracellular domain of wild type CTLA4 with amino acidchanges A29Y (a tyrosine amino acid residue substituting for an alanineat position 29) and L104E (a glutamic acid amino acid residuesubstituting for a leucine at position +104) joined to an Ig tail. FIG.20 illustrates L104EA29Ylg. The amino acid sequence of L104EA29Ylgcomprises alanine at amino acid position −1 to lysine at amino acidposition +357 as shown in FIG. 20. Alternatively, the amino acidsequence of L104EA29Ylg comprises methionine at amino acid position +1to lysine at amino acid position +357 as shown in FIG. 20. L104EA29Ylgcomprises a junction amino acid residue glutamine at position +125 andan Ig portion encompassing glutamic acid at position +126 through lysineat position +357. DNA encoding L104EA29Ylg was deposited on Jun. 20,2000, in the American Type Culture Collection (ATCC) under theprovisions of the Budapest Treaty, and has been accorded ATCC accessionnumber PTA-2104. 104EA29Y-Ig is described in co-pending U.S. patentapplication Ser. Nos. 09/579,927, 60/287,576 and 60/214,065, and inWO/01/923337 A2, which are incorporated by reference herein in theirentireties. The soluble L104EA29Ylg molecules produced by the culturingmethods of this invention may or may not include a signal (leader)peptide sequence. Typically, the molecules produced according to theinvention do not include a signal peptide sequence.

As used herein, the term “soluble” refers to any molecule, or fragmentthereof, not bound or attached to a cell, i.e., circulating. Forexample, CTLA4, can be made soluble by attaching an Ig moiety to theextracellular domain of CTLA4. Alternatively, a molecule such as CTLA4can be rendered soluble by removing its transmembrane domain. Typically,the soluble molecules produced according to the invention do not includea signal (or leader) sequence.

A soluble CTLA4 molecule refers to a non-cell-surface-bound (i.e.,circulating) molecule comprising wild type CTLA4, or any portion orderivative that binds B7, including, but not limited to, soluble CTLA4fusion proteins; soluble CTLA4 fusion proteins such as CTLA4lg fusionproteins (e.g., ATCC 68629), wherein the extracellular domain of CTLA4is fused to an Ig moiety that is all or a portion of an Ig molecule,preferably all or a portion of an Ig constant region, such as all or aportion of IgCγ1 (IgCgamma1), IgCγ2 (IgCgamma2), IgCγ3 (IgCgamma3),IgCγ4 IgCgamma4), IgCμ (IgCmu), IgCα1 (IgCalpha1), IgCα2 (IgCalpha2),IgCδ

(IgCdelta) or IgCε (IgCepsilon), rendering the fusion molecule soluble;soluble CTLA4 fusion proteins in which the extracellular domain is fusedor joined with a portion of a biologically active or chemically activeprotein such as the papillomavirus E7 gene product (CTLA4-E7),melanoma-associated antigen p97 (CTLA4-p97) or HIV env protein(CTLA4-env gp120), as described in U.S. Pat. No. 5,844,095, hereinincorporated by reference in its entirety; hybrid (chimeric) fusionproteins such as CD28/CTLA4lg as described in U.S. Pat. No. 5,434,131,herein incorporated by reference in its entirety; CTLA4 molecules withthe transmembrane domain removed to render the protein soluble (See,e.g., M. K. Oaks et al., 2000, Cellular Immunology, 201:144-153, hereinincorporated by reference in its entirety); the soluble CTLA4 mutantmolecule L104EA29Ylg.

A soluble CTLA4 molecule can also be a soluble CTLA4 mutant molecule.The soluble CTLA4 molecules produced according to this invention may ormay not include a signal (leader) peptide sequence. The signal peptidecan be any sequence that will permit secretion of the molecule,including the signal peptide from oncostatin M (Malik et al., 1989,Molec. Biol., 9:2847-2853), or CD5 (N. H. Jones et al., 1986, Nature,323:346-349), or the signal peptide from any extracellular protein. Thesoluble CTLA4 molecule produced by the culturing processes of theinvention can include the oncostatin M signal peptide linked at theN-terminal end of the extracellular domain of CTLA4. Typically, in theinvention the molecules do not include a signal peptide sequence.

“CTLA4 fusion protein” as used herein refers to a molecule comprisingthe extracellular domain of wild type CTLA4, or portion thereof thatbinds to B7, fused to a non-CTLA4 moiety that renders the CTLA4 moleculesoluble, such as an Ig moiety. For example, a CTLA4 fusion protein caninclude the extracellular domain of CTLA4 fused to all or a portion ofan Ig constant region. Examples of Ig constant domains (or portionsthereof) that may be fused to CTLA4 include all, but are not limited tothose listed hereinabove. A CTLA4 fusion protein can also be a CTLA4mutant molecule.

As used herein, “non-CTLA4 moiety” refers to a molecule or portionthereof that does not bind CD80 and/or CD86 and does not interfere withthe binding of CTLA4 to its ligand. Examples include, but are notlimited to, an Ig moiety that is all or a portion of an Ig molecule, aportion of a biologically active or chemically active protein such asthe papillomavirus E7 gene product (CTLA4-E7), melanoma-associatedantigen p97 (CTLA4-p97) or HIV env protein (CTLA4-env gp120) (asdescribed in U.S. Pat. No. 5,844,095, herein incorporated by referencein its entirety). Examples of Ig moieties include all or a portion of animmunoglobulin constant domain, such as IgCγ1 (IgCgamma1), IgCγ2(IgCgamma2), IgCγ3 (IgCgamma3), IgCγ4 IgCgamma4), IgCμ (IgCmu), IgCα1(IgCalpha1), IgCα2 (IgCalpha2), IgCδ

(IgCdelta) or IgCε (IgCepsilon). The Ig moiety can include the hinge,CH2 and CH3 domains, or the CH1, hinge, CH2 and CH3 domains of theaforementioned constant regions or other constant regions. Preferably,the Ig moiety is human or monkey and includes the hinge, CH2 and CH3domains. Most preferably the Ig moiety includes the hinge, CH2 and CH3domains of human IgCγ1, or is the hinge, CH2 and CH3 domains of humanIgCγ1. In an Ig moiety, the Ig constant region or portion thereof can bemutated so as to reduce its effector functions (see, e.g., U.S. Pat.Nos. 5,637,481, 5,844,095 and 5,434,131).

The extracellular domain of CTLA4 refers to any portion of wild typeCTLA4 that recognizes and binds B7. For example, an extracellular domainof CTLA4 comprises methionine at position +1 to aspartic acid atposition +124 (FIG. 21). For example, an extracellular domain of CTLA4comprises alanine at position −1 to aspartic acid at position +124 (FIG.21).

As used herein, the term “mutation” refers to a change in the nucleotideor amino acid sequence of a wild type molecule, for example, a change inthe DNA and/or amino acid sequences of the wild type CTLA4 extracellulardomain. A mutation in the DNA may change a codon leading to a change inthe encoded amino acid sequence. A DNA change may include substitutions,deletions, insertions, alternative splicing, or truncations. An aminoacid change may include substitutions, deletions, insertions, additions,truncations, or processing or cleavage errors of the protein.Alternatively, mutations in a nucleotide sequence may result in a silentmutation in the amino acid sequence, as is well understood in the art.As is also understood, certain nucleotide codons encode the same aminoacid. Examples include nucleotide codons CGU, CGG, CGC, and CGA whichencode the amino acid, arginine (R); or codons GAU, and GAC which encodethe amino acid, aspartic acid (D).

Thus, a protein can be encoded by one or more nucleic acid moleculesthat differ in their specific nucleotide sequence, but still encodeprotein molecules having identical sequences. The mutant molecule mayhave one, or more than one, mutation. For guidance, the amino acidcoding sequence is as follows:

One Amino Letter Acid Symbol Symbol Codons Alanine Ala A GCU, GCC, GCA,GCG Cysteine Cys C UGU, UGC Aspartic Acid Asp D GAU, GAC Glutamic AcidGlu E GAA, GAG Phenylalanine Phe F UUU, UUC Glycine Gly G GGU, GGC, GGA,GGG Histidine His H CAU, CAC Isoleucine Ile I AUU, AUC, AUA Lysine Lys KAAA, AAG Leucine Leu L UUA, UUG, CUU, CUC, CUA, CUG Methionine Met M AUGAsparagine Asn N AAU, AAC Proline Pro P CCU, CCC, CCA, CCG Glutamine GlnQ CAA, CAG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Serine Ser S UCU,UCC, UCA, UCG, AGU, AGC Threonine Thr T ACU, ACC, ACA, ACG Valine Val VGUU, GUC, GUA, GUG Tryptophan Trp W UGG Tyrosine Tyr Y UAU, UAC

As used herein, a “fragment or portion” is any part or segment of amolecule. For CTLA4 or CD28, a fragment or portion is preferably theextracellular domain of CTLA4 or CD28, or a part or segment thereof,that recognizes and binds B7 or interferes with a B7 so that it blocksbinding to CD28 and/or CTLA4. Also, as used herein, “corresponding”means sharing sequence identity.

As used herein, a “derivative” is a molecule that shares sequencesimilarity and activity of its parent molecule. For example, aderivative of CTLA4 includes a soluble CTLA4 molecule having an aminoacid sequence at least 70% similar to the extracellular domain ofwildtype CTLA4, and which recognizes and binds B7 e.g. CTLA4lg orsoluble CTLA4 mutant molecule L104EA29Ylg. A derivative means any changeto the amino acid sequence and/or chemical quality of the amino acide.g., amino acid analogs.

As used herein, to “regulate an immune response” is to activate,stimulate, up-regulate, inhibit, block, reduce, attenuate, down-regulateor modify the immune response. A variety of diseases, e.g., autoimmunediseases, may be treated by regulating an immune response, e.g., byregulating functional CTLA4- and/or CD28-positive cell interactions withB7-positive cells. For example, a method of regulating an immuneresponse comprises contacting B7-positive cells with a soluble CTLA4molecule, such as those produced according to this invention, to formsoluble CTLA4/B7 complexes, wherein the soluble CTLA4 moleculeinterferes with the reaction of an endogenous CTLA4 and/or CD28 moleculewith the B7 molecule. To “block” or “inhibit” a receptor, signal ormolecule, as referred to herein, means to interfere with the activationof the receptor, signal or molecule, as detected by an art-recognizedtest. Blockage or inhibition can be partial or total.

As used herein, “blocking B7 interaction” refers to interfering with thebinding of B7 to its ligands, such as CD28 and/or CTLA4, therebyobstructing T-cell and B7-positive cell interactions. Examples of agentsthat block B7 interactions include, but are not limited to, moleculessuch as an antibody (or portion thereof) that recognizes and binds tothe any of CTLA4, CD28 or B7 molecules (e.g., B7-1, B7-2); a solubleform (or portion thereof) of the molecules such as soluble CTLA4; apeptide fragment or other small molecule designed to interfere with thecell signal through a CTLA4/CD28/B7-mediated interaction. In a preferredembodiment, the blocking agent is a soluble CTLA4 molecule, such asCTLA4lg (ATCC 68629) or L104EA29Ylg (ATCC PTA-2104); a soluble CD28molecule, such as CD28lg (ATCC 68628); a soluble B7 molecule, such asB7-Ig (ATCC 68627); an anti-B7 monoclonal antibody (e.g., ATCC HB-253,ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341 and monoclonalantibodies as described in U.S. Pat. No. 6,113,898 or in Yokochi et al.,1982, J. Immunol., 128(2):823-827); an anti-CTLA4 monoclonal antibody(e.g., ATCC HB-304, and monoclonal antibodies as described in references82-83); and/or an anti-CD28 monoclonal antibody (e.g. ATCC HB 11944 andMAb 9.3, as described in Hansen et al., 1980, Immunogenetics, 10:247-260, or Martin et al., 1984, J. Clin. Immunol., 4(1):18-22).Blocking B7 interactions can be detected by art-recognized tests such asdetermining reduction of immune disease (e.g., rheumatic disease)associated symptoms, by determining a reduction in T-cell/B7-cellinteractions, or by determining a reduction in the interaction of B7with CTLA4/CD28. Blockage can be partial or total.

As used herein, an effective amount of a molecule refers to an amountthat blocks the interaction of the molecule with its ligand. Forexample, an effective amount of a molecule that blocks the interactionof B7 with CTLA4 and/or CD28 is the amount of the molecule that, whenbound to B7 molecules on B7-positive cells, inhibits B7 molecules frombinding endogenous ligands such as CTLA4 and CD28. Alternatively, aneffective amount of a molecule that blocks the interaction of B7 withCTLA4 and/or CD28 is the amount of the molecule that, when bound toCTLA4 and/or CD28 molecules on T cells, inhibits B7 molecules frombinding endogenous ligands such as CTLA4 and CD28. The inhibition orblockage can be partial or complete.

For clinical protocols, it is preferred that the Ig moiety of a fusionprotein, such as CTLA4lg or mutant CTLA4lg, does not elicit adetrimental immune response in a subject. The preferred moiety is all ora portion of the Ig constant region, including human or non-humanprimate Ig constant regions. Examples of suitable Ig regions includeIgCγ1 (IgCgamma1), IgCγ2 (IgCgamma2), IgCγ3 (IgCgamma3), IgCγ4IgCgamma4), IgCμ (IgCmu), IgCα1 (IgCalpha1), IgCα2 (IgCalpha2), IgCδ

(IgCdelta) or IgCε (IgCepsilon), including the hinge, CH2 and CH3domains, or the CH1, hinge, CH2 and CH3 domains, which are involved ineffector functions such as binding to Fc receptors, complement-dependentcytotoxicity (CDC), or antibody-dependent cell-mediated cytotoxicity(ADCC). The Ig moiety can have one or more mutations therein, (e.g., inthe CH2 domain to reduce effector functions such as CDC or ADCC) wherethe mutation modulates the capability of the Ig to bind its ligand byincreasing or decreasing the capability of the Ig to bind to Fcreceptors. For example, mutations in the Ig moiety can include changesin any or all of its cysteine residues within the hinge domain. Forexample, the cysteines at positions +130, +136, and +139 are substitutedwith serine. The Ig moiety can also include the proline at position +148substituted with a serine. Further, mutations in the Ig moiety caninclude having the leucine at position +144 substituted withphenylalanine; leucine at position +145 substituted with glutamic acid;or glycine at position +147 substituted with alanine.

EXAMPLES

The following Examples set forth specific aspects of the invention toillustrate the invention and provide a description of the presentmethods for those of skill in the art. The Examples should not beconstrued as limiting the invention, as the Examples merely providespecific methodology and exemplification that are useful in theunderstanding and practice of the invention and its various aspects.

Examples 1-5 as set forth below describe experiments relating to cellculture processes involving addition of glucocorticoids during theculture run.

Example 1

In this study, the intracellular effects of dexamethasone (DEX) on theCHO cell glycosylation process, and the extracellular effects due tosialidase activity were studied. Here it is demonstrated for the firsttime that DEX was capable of improving the sialylation of a recombinantfusion glycoprotein produced by CHO. The net effects of DEX in promotingincreased sialylation tested in shake flask cultures were thensuccessfully confirmed in controlled bioreactors, and resulted inenhanced sialic acid content as well as reduced de-sialylation rates inthe fed-batch cultures.

Cell Line and Medium

The CHO cell line used in this study was originally subcloned from DG44parental cells and cultured in a proprietary protein-free growth medium.

Shake Flask Experiments

The experiments were carried out in 250-mL shake flasks (VWRinternational) with starting volumes of 100 mL and initial celldensities of 6×10⁵ cells/mL. The cultures were placed on a shakerplatform (VWR international) at 150 rpm and maintained at 37° C. and 6%CO₂ for ten days. The cultures were sampled daily and the pH wasadjusted as needed using 1M sodium carbonate and fed with glucose andglutamine every two days in order to maintain them at adequate levels.Cell density and viability were measured offline using a Cedex automatedcell counter (Innovatis AG, Bielefeld, Germany). Culture pH andconcentrations for glucose and glutamine were measured off-line using aBioprofile Analyzer 400 (Nova Biomedical Corporation, Waltham, Mass.).

Bioreactor Operation

Bioreactor experiments were performed in 5-L bioreactors (Sartorius AG,Goettingen, Germany) with initial working volumes of 1.5 L. Agitation,pH, and dissolved oxygen were controlled at 150 rpm, 7.05, and 50% airsaturation, respectively. Temperature was initially controlled at 37°C., but was shifted to a lower temperature during the culture to extendculture viability. The bioreactors were operated in fed-batch mode andwere fed daily starting on day 3 with the proprietary feed medium tomaintain adequate concentrations for glucose and other nutrients.Samples were taken during the cell culture process and analyzed for celldensity, cell viability, substrates and metabolites.

Western Blot Analysis of α2,3-Sialytransferase (α2,3-ST) andβ1,4-Galactosyltransferase (β1,4-GT)

Approximately 10⁷ CHO cells were washed with 1× Phosphate BufferedSaline (PBS), lysed with 1 mL of Laemmli sample buffer (Bio-RadLaboratories, Hercules, Calif.), then denatured at 90° C. for 5 minutes.The whole cell lysates were separated on a 4-15% SDS-polyacrylamide gel,blotted to 0.45 μm nitrocellulose membranes (Bio-Rad Laboratories,Hercules, Calif.), and probed with primary and secondary antibodies. Theprimary antibodies were anti-human α2,3-ST rabbit polyclonal antibodiesand anti-human β1,4-GT rabbit polyclonal antibodies (Santa CruzBiotechnology, Santa Cruz, Calif.). The secondary antibody washorseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Santa CruzBiotechnology, Santa Cruz, Calif.). The membranes were stripped andre-probed with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz,Calif.) and an HRP-conjugated anti-mouse secondary antibody.Immunodetection was performed using an enhanced chemiluminescenceWestern blotting detection system (GE Healthcare) and visualized with aVersaDoc Imaging System (Bio-Rad Laboratories, Hercules, Calif.).

Measurement of Sialidase Activity in Supernatant

Sialidase activity was analyzed using the Amplex Red Neuraminidase(Sialidase) Assay Kit (Invitrogen, Carlsbad, Calif.). Briefly,neuraminidase in the sample is used to desialylate fetuin. This assayutilizes Amplex Red to detect H₂O₂ generated by oxidation of thedesialylated galactose residues on the fetuin by galactose oxidase. TheH₂O₂, in the presence of HRP, reacts stoichiometrically with Amplex Redreagent to generate the red-fluorescent oxidation product, resorufin 5,which is then analyzed either fluorometrically orspectrophotometrically. In each assay, 50 μL of working solution (100 μMAmplex Red, 0.2 U/mL HRP, 4 U/mL galactose oxidase and 500 μg/mL fetuin)was added into each microplate well containing 50 μL of diluted cellculture supernatant. After 30 minutes incubation at 37° C., samples wereanalyzed for absorbance at 560 nm using a microplate reader.

Sialic Acid Assay

The sialic acid to recombinant protein molar ratio was calculated bydetermining the sialic acid and recombinant protein concentrations.Sialic acid (SA), including N-Acetylneuraminic (Neu5Ac) andN-glycolylneuraminic acid (Neu5Gc), was released by partial acidhydrolysis. The derivatives were then separated by reversed-phase HPLCto determine sialic acid content. The protein concentrations weredetermined by UV absorbance at 280 nm. Sialic acid content is reportedas molar ratios, which is the total moles of Neu5Ac and Neu5Gc per moleof recombinant glycoprotein. Sialic acid contents are reported asnormalized values, which are the actual values divided by an arbitraryvalue. The same divisor was used to normalize all sialic acid contentdata in the studies reported here.

N-Linked Oligosaccharides Measurement

High-pH anion-exchange chromatography with pulsed amperometric detection(HPAEC-PAD) was used for the profiling of N-linked oligosaccharides(Base and Spellman 1990; Townsend and Hardy 1991) to provide informationon site-specific N-linked glycosylation. N-linked oligosaccharides werecleaved from protein and separated on HPAEC-PAD into four domains basedon the amount of sialic acid present. Domain I represents the asialospecies. Domains II, III, and IV are sialylated species and representmonosialylated, disialylated, and tri- and tetrasialylated species,respectively. N-linked sialylated species fraction is reported as thepercentage of N-linked sialylated species within the total N-linkedoligosaccharides species, which includes both asialo and sialylatedspecies. N-linked sialylated species fractions are reported asnormalized values, which are the actual values divided by an arbitraryvalue. The same divisor was used to normalize all N-linked sialylatedspecies fraction data in the studies reported here.

O-Linked Oligosaccharides Measurement

O-linked glycosylation was characterized by intact mass analysis of areference standard and Protein-A purified samples of the fusion protein(Reference). Samples were diluted in 100 mM Tris, 25 mM NaCl, pH 7.6 andincubated with PNGase F overnight to enzymatically remove all N-linkedoligosaccharides. Samples were then injected for intact mass analysisafter mixing with an internal standard of insulin. O-linked sialic acidcontent is reported as molar ratios, which is the moles of O-linkedsialic acid per mole of recombinant glycoprotein.

Results

Dexamethasone Enhances Sialic Acid Content and Percentage of SialylatedSpecies in N-Linked Oligosaccharides

The CHO DG44 cell line expressing the fusion glycoprotein was treatedwith various levels of DEX to assess whether DEX affected sialic acidcontent and the percentages of the different sialylated species. Thisstudy was performed in duplicate 250-mL shake flasks using the cultureconditions as described above. On the second day after inoculation, DEXwas added to CHO cell cultures at final concentrations ranging from 0.01to 10 μM DEX. Cultures were harvested on day 10, and the fusion proteinwas purified using a protein A column and analyzed for total sialic acidcontent, O-linked sialic acid content and the N-linked profile.Sialylation increased in the DEX-treated cultures in aconcentration-dependent manner (Table 1).

TABLE 1 DEX Percent increase Percent increase Percent increase Percentincrease Percent increase treatment Percent increase of N-link ofN-link- of N-link- of N-link- of O-link (μM) of SA (%) asialo- (%) Mono-(%) Di- (%) Tri and tetra- (%) SA (%) 0.01 9.3 −8.6 6.6 13.9 4.5 0.0 0.113.0 −9.6 7.3 13.9 13.6 0.5 1.0 13.0 −10.0 4.3 19.4 15.9 0.9 10 20.4−15.6 7.3 24.3 20.5 0.0

DEX concentrations from 0.01 μM to 10 μM resulted in 9.3% to 20.4%increase in the sialic acid content, with a maximal effect at 10 μM.Compared with the control, the N-linked oligosaccharide chromatographsfor cultures with DEX treatment showed enhanced monosialylated,disialylated and the tri-plus tetra sialylated fractions of 4.3% to7.3%, 13.9% to 24.3% and 4.5% to 20.5%, respectively. Conversely, therewas a 8.5% to 15.6% reduction compared to the control in asialofractions in the oligosaccharide distributions for the DEX-supplementedcultures. The N-linked chromatograms also showed the maximum effect at10 μM DEX. These results indicate improved sialylation in the DEXtreated cultures. However, no significant changes were observed in theO-linked sialic acid molar ratios from the DEX treated samples.

DEX Promotes β1,4-Galactosyltransferase (β1,4-GT) andα2,3-Sialyltransferase (α2,3-ST) Expression

Western blotting was employed to elucidate potential mechanisms of DEXon sialylation by studying the expression of two enzymes, β1,4-GT andα2,3-ST, which are involved in the sialic acid addition pathways. Inthis experiment, DEX was added on the second day after inoculation tocultures at concentrations from 0.1 μM to 10 μM and the cells wereharvested for Western blot analysis after three days. The housekeeperprotein β-actin was used to compare sample loading. As shown in FIGS. 1Aand 1B, the expression of β1,4-GT and α2,3-ST levels increasedsubstantially in the DEX treated cultures compared to cultures withoutDEX treatment. Expression intensity for both enzymes generally increasedwith the DEX concentration. These results demonstrated that DEX wascapable of stimulating expression of the sialyltransferases β1,4-GT andα2,3-ST in CHO cells.

Cell Protective Effect of DEX Results in Reduced Sialidase Activity inthe Culture Supernatant

In Example 3, DEX treatment was shown to increase expression of theanti-apoptotic protein GILZ resulting in enhanced viability of the CHOcell cultures. To determine whether the improvement in cell viabilitydue to DEX could lessen the degradative effect of sialidases on therecombinant fusion protein, a shake flask study was initiated to comparecell viability and supernatant sialidase activity profiles from cultureswith and without 1 μM DEX. As shown in FIG. 2A and FIG. 2B, increasedsialidase activity was associated with the decreased cell viabilities inboth the DEX-treated and untreated cultures. Cell viability decreasedfrom 98.0±0.1% on day 4 to 85.0±2.7% on day 10 in the DEX treatedcultures compared to a day 10 viability of 70.2±4.6% for the control.The absorbance measurement of sialidase activity increased from0.006±0.002 on day 4 to 0.024±0.003 on day 10 in the DEX treatedcultures compared to a day 10 value of 0.047±0.004 in the control. Thus,the rate at which the cultures both declined in cell viability as wellas increased in sialidase activity were significantly slower in theDEX-treated cultures. These results suggest that DEX was capable ofinhibiting sialidase release through its cell protective effect.

Comparison of Dexamethasone with Other Two Glucocorticoids,Hydrocortisone and Prednisolone

Additional glucocorticoid compounds, hydrocortisone (HYC) andprednisolone (PRD) were also evaluated to determine whether the effectof DEX on increasing sialylation in CHO cells could be extended to otherglucocorticoid compounds. DEX, HYC and PRD were added into cell culturemedium on the second day after inoculation at the final concentrationsof 0, 0.1, 1 and 10 μM. FIG. 3A and FIG. 3B show the normalized totalsialic acid content and normalized N-linked sialylated species fractionafter the 10-day culture for the different glucocorticoid conditions.The total sialic acid content at glucocorticoid concentrations between0.1 to 10

M were 12.4±0.4 to 14.0±0.5 for DEX, 11.2±0.1 to 13.0±0.2 for HYC, and11.8±0.2 to 13.4±0.8 for PRD, compared to 10.2±0.2 for the control. TheN-linked sialylated species fraction at glucocorticoid concentrationsbetween 0.1 to 10

M were 85.6±1.6 to 88.8±0.9 for DEX, 79.8±1.6 to 89.6±0.1 for HYC, and83.7±0.1 to 89.0±0.2 for PRD compared to 77.1±0.7 for the control. Thus,similar to DEX, both HYC and PRD also showed increases in sialylationand the maximum effect was observed at 10 μM for all threeglucocorticoid compounds within the studied concentration range.However, higher concentrations of HYC and PRD were required to achievethe same level of sialylation enhancement as DEX.

Mechanism for Sialylation Enhancement by Dexamethasone Involves theGlucocorticoid Receptor.

In order to determine whether the sialylation improvement from DEXaddition was mediated through the glucocorticoid receptor (GR), the GRantagonist mifepristone (RU-486) was added to cell culture medium beforeDEX treatment. Specifically, 1 μM of RU-486 was added 48 hours afterinoculation and DEX, at concentrations of 0.1, 1 and 10 μM, was thenadded 24 hours later. DEX was also added to cultures without RU-486 ascontrols. For conditions with and without RU-486, the normalized totalsialic acid content and normalized N-linked sialylated species fractioninduced by DEX are shown in FIG. 4A and FIG. 4B, respectively. Theability of DEX to enhance product sialylation was substantially reducedin the presence of 1 μM of RU-486 and was most evident for the 0.1 μMDEX condition. The total sialic acid content and N-linked sialylatedspecies fraction for the 0.1 μM DEX conditions decreased from 15.1±0.1and 92.2±2.0 (without RU-486) to 12.7±0.1 and 81.5±0.8 (with 1 μMRU-486), respectively. These results indicate that the mechanism throughwhich DEX increased sialylation was GR-dependent, since RU-486 competeswith DEX for the ligand-binding domain of the GR (Raux-Demay et al.1990).

Application of DEX in Fed Batch Bioreactor Culture

The effect of DEX to increase sialylation of fusion protein found inshake flasks was then tested in fed-batch cultures using controlled 5-Lbioreactors. The bioreactors were operated as described in the methodssection. Higher total sialic acid content (FIG. 5A) and N-linkedsialylated species fraction (FIG. 5B) were observed in the cultures with1 μM DEX bolus addition. The total sialic acid content with DEX was16.5±0.1 compared to 14.2±0.1 for the control (16.2% increase).Similarly, the N-linked sialylated species fraction with DEX was90.2±1.3 compared to 77.9±1.6 for the control (15.8% increase). Inagreement with the observation in shake flask sialidase activity studies(FIGS. 3A and 3B), the total sialic contents decreased between days 8 to14 from 17.9±0.1 to 16.5±0.1 (−7.8%) with DEX compared 16.3±0.1 to14.2±0.1 (−12.9%) for the control. Similarly, the percentage of thesialylated fraction between days 8 to 14 decreased from 97.7±0.9 to90.2±1.3 (−7.7%) with DEX compared to 91.3±0.9 to 77.9±1.6 (−14.7%) forthe control.

The increase in glycoprotein sialylation by DEX was further confirmed atvarious bioreactor scales. The normalized final sialic acid content, andnormalized percentage of sialylated fractions from different runs aresummarized in FIG. 6. Within these runs, the DEX condition included runswith DEX added as a bolus or included in the feed medium. As indicatedin FIG. 6, DEX showed significant sialyaltion improvement with enhancedfinal sialic acid content (p value <0.001 by t-test) and finalsialylated species fraction (p value <0.001 by t-test).

Conclusion

The addition of dexamethasone to a CHO culture producing a recombinantfusion glycoprotein resulted in improved sialylation. This study was thefirst to demonstrate that dexamethasone was capable of increasingexpression of the glycosyltransferases α2,3-ST and β1,4-GT and that theeffect of dexamethasone was mediated through the glucocorticoidreceptors. Overall the effect of dexamethasone in improving sialylationinvolved both intracellular effects through the glycosylatransferases aswell as extracellular effects through extending culture viability whichdecreased the presence of sialidases released into the culturesupernatant through cell lysis. Dexamethasone was found to be aconvenient method for improvement of sialylation.

Example 2

Cell Line and Medium

The CHO cell line used in this study was originally subcloned from DG44parental cells and cultured in a protein-free proprietary growth medium.The host cells were genetically engineered to secrete an IgG-fusionprotein under the control of CMV promoter.

Shake Flask Experiments

The experiments were carried out in 250-mL shake flasks (VWRinternational) with starting volumes of 100 mL and initial celldensities of 6×10⁵ cells/mL. Shake flasks were placed on a shakerplatform (VWR international) at 150 rpm rotating speed. Cells werecultured under standard humidified condition at 37° C. and 6% CO₂, for aten-day duration with daily pH adjustment using 1M sodium carbonate, andfed with glucose and glutamine every two days in order to maintain themat certain level. Cell density, cell viability andsubstrates/metabolites were offline-analyzed by an automated cellcounting system Cedex (Innovatis AG, Bielefeld, Germany) and aBioprofile Analyzer 400 (Nova Biomedical Corporation, Waltham, Mass.).Supernatant from each culture harvest was collected for SEC analysis.

Size-Exclusion Chromatography (SEC)

SEC analysis was performed according to the previously published method(Perico et al., 2008) with some modifications. Briefly, the Protein-Apurified samples were run on an Agilent 1100 HPLC system (AgilentTechnologies, Inc., Palo Alto, Calif.) on Tosoh Bioscience TSK-Gel G3000SWxl column (7.8 ID×30 cm, 5 um particles). The mobile phase contained1×Phosphate Buffered Saline (PBS) at pH 7.4. The flow rate was 0.5mL/min, and column temperature was controlled at 25° C. The signal wasmonitored by absorbance at wavelength of 280 nm.

Western Blot Analysis of Glutathione Reductase and GlucocorticoidReceptor

After being washed with 1×PBS, approximately 10⁷ CHO cells were lysedwith 1 mL of Laemmli sample buffer (Bio-Rad Laboratories) and denaturedat 90° C. for 5 minutes. The whole cell lysates were separated on a4-15% SDS-polyacrylamide gel, blotted to 0.45 μm nitrocellulosemembranes (Bio-Rad Laboratories), and probed with primary antibodies andsecondary antibody. The primary antibody for detection of glutathionereductase was the monoclonal antibody raised against amino acids 391-510mapping near the C-terminus of glutathione reductase of human origin(Santa Cruz Biotechnology, Santa Cruz, Calif.). Although Chinese hamsterorigin was not listed by the manufacturer for detection of glutathionereductase using this antibody, a preliminary experiment showed thatthere was only one band detected in the lysates from both CHO cells andhuman origin HL60 cells with an identical apparent molecular size on theblot. The primary antibody used for detection of glucocorticoid receptorwas anti-human monoclonal antibody (Santa Cruz Biotechnology, SantaCruz, Calif.). The secondary antibody was horseradish peroxidase(HRP)-conjugated anti-mouse antibody (Santa Cruz Biotechnology, SantaCruz, Calif.). Membranes were stripped and re-probed with β-actinantibody (Santa Cruz Biotechnology) and HRP-conjugated secondaryantibody. Immunodetection was performed with the enhancedchemiluminescence Western blotting detection system (GE Healthcare) andvisualized with VersaDoc Imaging System (Bio-Rad Laboratories).

Results

Dexamethasone (DEX) Reduces Aggregation of CHO-Produced IgG-FusionProteins at Broad Range of Concentrations

FIG. 7A shows the significantly reduced percentage of high molecularweight (HMW) species in IgG-fusion protein after the CHO cells weretreated with DEX at 1 μM final concentration in culture medium. The maincomponents of HMW species were dimers and trimers formed covalently ornon-covalently and categorically considered to be protein aggregates.Considering that a 15% reduction of protein aggregation was obtainedunder the culture conditions that had been already optimized, theimprovement was practically significant in manufacture process. Toestablish the dose-response curve and time-course for the effects of DEXon protein aggregation, we cultured the cells either at differentconcentration of DEX or at the same concentration (1 μM) but withdifferent incubation time. As shown in FIG. 7C, DEX time-dependentlyreduced the rate of protein aggregation, which was consistent withpreviously observed time-dependency of DEX on the improvement of cellviability and protein glycosylation profiles (Table 1 and FIG. 11).However, the concentration-dependency of DEX on reduction of proteinaggregation level was not obvious (FIG. 7B), which suggests that theanti-aggregation effect was not simply due to the improved cellviability because our previous results demonstrated the viability of CHOcells was increased with the concentration of DEX from 0.01 μM to 10 μMin the culture medium

Dexamethasone Up-Regulates Glutathione Reductase Expression in CHO Cells

A Western Blot was performed to detect glutathione reductase expressionin the whole cell lysates prepared from the CHO cells treated withvarious concentrations of DEX. As shown in FIG. 8, DEX increased theexpression of glutathione reductase, which could be seen even when thedrug was at 1 nM. Detection of β-actin was used for sample loadingcomparison to eliminate the possibility that the glutathione reductaseband enhancement was due to the coincidently increased sample loading.

GSH Reduces Purified IgG-Fusion Protein Aggregation In Vitro

To determine if GSH itself could affect protein aggregation by directlyinteracting with the proteins, we did in vitro study by adding GSH toProtein-A purified IgG-fusion proteins that were reconstituted inTris-acetate buffer, and analyzed SEC profiles. As shown in FIG. 9, thepercentage of high molecular weight species was significantly decreased,with the reduction rate being 15.3% and 27.3% in the presence of 1 and 3mM GSH respectively. The data clearly demonstrated that GSH coulddirectly inhibit IgG-fusion protein aggregation.

Dexamethasone Effects are Mediated Via Glucocorticoid Receptors

DEX is a potent glucocorticoid with broad pharmacological actions. Todetermine if the inhibitory effect of DEX on IgG-fusion proteinaggregation was specifically mediated through activation ofglucocorticoid receptors, it was first confirmed that there isendogenous expression of glucocorticoid receptors (GR) in the cell lineutilized, which is presented in FIG. 10A. Since the antibody used in theWestern Blotting was raised against the conserved region of human GR, awhole-cell lysate sample of HepG-2 cells was also loaded as human originsample for antibody validation purpose. Although the antibody coulddetect both GRα and GRβ, the present analysis only showed a single band.This could be because the molecular weights of these two isoforms (95kDa vs 90 kDa) were too close to be separated on the 5-15% gradient gelor more likely because there was only one isoform presented in thesamples.

Since DEX was found to up-regulate glutathione reductase expression inCHO cells (see FIG. 8), a further assessment to determine if this effectwas induced by the activation of GR receptors. RU-486 is a GR antagonistand often used as a tool drug in many GR related studies. Ourpreliminary experiment showed that RU-486 did not affect the CHO cellviability and metabolic parameters if the concentrations were not higherthan 1 μM, at 10 μM, a significant decrease in cell viability and cellgrowth rate was observed. In the subsequent experiments, RU-486 wastherefore used at 1 μM or lower concentrations and it was introducedinto the culture to pre-treat CHO cells one day before DEX addition.FIG. 10B shows that in the presence of 1 μM RU-486, the up-regulatoryeffect of DEX on glutathione reducatase expression was attenuated,confirming the involvement of GR.

Finally, the percentages of HMW species in IgG-fusion proteins producedby CHO cells treated with 0.1 μM DEX and its combination with differentconcentrations of RU-486 was evaluated. Consistent with the result inFIG. 7B, the percentage of HMW species was decreased by approximately17% when the culture was treated with 0.1 μM DEX alone, and this effectwas gradually diminished with increasing concentrations of RU-486 (FIG.10C). Under the condition that equal concentration of DEX and RU-486 wasused, the inhibition rate of HMW species was about half of the rateobtained from DEX alone, indicating that DEX as an agonist and RU-486 asan antagonist had similar affinities to compete with each other for GRin CHO cells. Thus, taken all three pieces of data in FIG. 9 together,the results clearly demonstrated that the inhibition of IgG-fusionprotein aggregation was mediated through GR.

Conclusion

As an inexpensive but potent glucocorticoid and with its beneficialtriad, improving cell viability as described in Example 3 andglycosylation as described in Example 1 and inhibiting proteinaggregation as described herein, dexamethasone may provide a simple,cost effective and efficient way to overall enhance cell culture process

Example 3

This study, for the first time, demonstrated that dexamethasone iscapable of preventing CHO cell apoptosis in serum-free condition in aconcentration- and time-dependent manner. DEX induces CHO cell GILZ(glucocorticoid-induced leucine zipper) gene expression. DEX wassuccessfully used in 10-L bioreactor CHO culture to improve cellviability and Fc-fusion protein productivity and sialic acid content.This study demonstrated the application of DEX in industrial cellculture to improve recombinant protein productivity and glycosylation.

Cell Line and Medium

The CHO cell line used in this study was subcloned from DG44 parentalcells and cultured in a proprietary chemically-defined growth medium.

Shake Flask Experiments

The experiments were carried out in 250-mL shake flasks (VWRinternational) with starting volumes of 100 mL and initial celldensities of 6×10⁵ cells/mL. The cultures were placed on a shakerplatform (VWR international) at 150 rpm and maintained at 37° C. and 6%CO₂ for ten days. The cultures were sampled daily and the pH adjustmentusing 1M sodium carbonate, and fed with glucose and glutamine every twodays in order to maintain them at certain levels. Cell density andviability were measured off-line a Bioprofile Analyzer 400 (NovaBiomedical Corporation, Waltham, Mass.).

Bioreactor Operation

Bioreactor experiments were performed in 10-L bioreactors (SartoriusStedim Biotech, France) with starting working volumes of 5 L. Agitation,pH and dissolved oxygen were controlled at 150 rpm, 7.05, and 50% airsaturation, respectively. Temperature was initially controlled at 37° C.and was shifted to lower temperature during the culture to extend theculture viability. The bioreactors were operated in a batch fed mode andwere fed daily starting on day 3 with the proprietary feed medium tomaintain adequate concentrations for glucose and other nutrients.Samples were taken during the cell culture process and analyzed for celldensity, cell viability, substrates and metabolites.

qRT-PCR Analysis

TaqMan® 5′-nuclease real-time quantitative RT-PCR assay was performed toverify the upregulation of GILZ by DEX. Total RNA, purified from eachtriplicate culture with and without 1 μM DEX using the RNeasy® midi kitas described above. Purified RNA was treated with RNasefree DNasel(Qiagen) and then used as a template to synthesize the first-strand cDNAusing RT² First Strand Kit (SA Biosciences, Frederick, Md.). Expressionof GILZ was quantitatively determined using GILZ specific TaqMan MGBprobe (6-FAM-AGAGGACTTCACGTGT) and primers (Forward:5-CCTCCCTCATCTGTCCACTGA-3 and Reverse: 5-TGGTGGGTTTGGCATTCAA-3). 20 ngof cDNA was amplified using 900 nM primers and 250 nM probe in 1× TaqManFast Universal PCR Master Mix (Applied Biosystems, Carlsbad, Calif.).Reactions were run in triplicate on the Applied Biosystems 7500 FastReal-Time PCR System using the universal cycling parameters (20 s 95°C., 40 cycles of 3 s 95° C., 30 s 60° C.). Parallel reactions were setup on the same plate analyzing β-Actin in each sample as an endogenouscontrol. The threshold cycle of each gene was normalized against thethreshold cycle of “housekeeping gene” β-Actin. Normalized changes ingene expression with respect to the control were calculated usingdelta-delta threshold cycle method (Livak and Schmittgen, 2001).

Western Blot Analysis of GILZ

Approximately 10⁷ CHO cells were washed with 1× phosphate bufferedsaline (PBS) solution, lysed with 1 mL of Laemmli sample buffer (Bio-RadLaboratories, Hercules, Calif.), and then denatured at 90° C. for 5minutes. The whole cell lysates were separated on a 4-15%SDS-polyacrylamide gel, blotted to 0.45 μm nitrocellulose membranes(Bio-Rad Laboratories), and probed with a primary mouse monocolonalantibody directed to GILZ (Santa Cruz Biotechnology, Santa Cruz,Calif.), followed by a horseradish peroxidase (HRP)-conjugatedanti-mouse secondary antibody (Santa Cruz Biotechnology). Membranes werestripped and re-probed with β-actin antibody (Santa Cruz Biotechnology)and an HRP-conjugated anti-mouse secondary antibody. Immunodetection wasperformed with the enhanced chemiluminescence Western blotting detectionsystem (GE Healthcare UK Limited Little Chalfont, Buckinghamshire, UK)and visualized with a VersaDoc Imaging System (Bio-Rad Laboratories,Hercules, Calif.).

Titer Assay

Titer was determined by affinity chromatography using an HPLC pump andUV detection (Agilent Technologies, Santa Clara, Calif.) and AppliedBiosystems Poros A/20 Protein A column (100×4.6 mm). The eluted proteinwas quantified using a 10-level standard curve.

Results Effect of Dexamethasone on CHO Cell Growth

The glucocorticoid dexamethasone (DEX) was added to shake flask culturesat final concentrations between 0.01 to 10 μM two day s afterinoculation to assess the effects of glucocorticoids on CH cellviability and the potential to extend the cell culture period. Theviable cell density (VCD) profile in the dose-response study (FIG. 11A)shows increasing inhibition of cell growth occurring one day after DEXtreatment in a dose-dependent manner. The peak VCD reached 8.5×10⁶cells/ml in the untreated control culture, whereas the peak VCD was6.5×10⁶ cell/mL in the cultures treated with 10 μM DEX. Cell viabilitydecreased rapidly after Day 6 (FIG. 11B) and percent viability on day 10was only 42.1%. In contrast, the final cell viabilities with 0.01 μM,and 10 μM DEX were 53.1% and 64.0%, respectively. The loss of viabilitybeginning on day 6 improved in a concentration-dependent manner in theDEX-treated cultures, with a maximal effect at 10 μM (FIGS. 11A and11B).

A time-course study for the addition of DEX was then performed in shakeflask cultures in which DEX was added to a final DEX concentration of 1μM and added between the day of inoculation through the sixth day afterinoculation. As shown in FIGS. 11C and 11D, the final VCD and cellviability increased in all DEX-treated cultures, and both VCD and cellviability improved in a time-dependent manner with earlier DEX additiontiming. The viability of untreated CHO cell cultures decreased to 50% onday 10, while cultures in which DEX was added on days 0, 2, 4 and 6 were63, 68, 72 and 74%, respectively.

DEX Reduces Cell Specific Growth Rate but Increases Cell SpecificProductivity

The specific growth rate, normalized volumetric productivity, andnormalized cell specific productivity were quantified and comparedbetween DEX-treated and untreated cells. The data comparing the specificcell growth rates and normalized productivity profiles with and without1 μM DEX are shown in FIGS. 12A and 12B (n=5). DEX was added on day 2,resulting in approximately a 30% reduction in cell growth rates with DEXcompared to control the day after DEX addition. However, this growthinhibition effect decreased with the culture time. At the end of theculture period, cell death rates were slower in cultures with DEXtreated cells. Moreover, the early cell growth inhibition induced by DEXdid not affect volumetric productivity (6.5 for all cultures); however,cultures with DEX treatment had significantly higher specificproductivity than untreated cells, with a maximum specific productivityof (7.0±0.6)×10⁻⁹.

Upregulation of GILZ by DEX was Confirmed by qRT-PCR and Western BlotAnalysis

GILZ was analyzed using qRT-PCR to validate the gene expression profilesobtained by microarray analysis. RNA samples from day 5 and 8 fortriplicate DEX treated and untreated cultures were applied in TaqMan®qPCR quantification. Parallel reactions were set up on the same plateanalyzing β-Actin in each sample as an endogenous control. As shown inFIG. 13A, the normalized expression profile changes of GILZ obtained byqRT-PCR on day 5 and day 8 samples are 7.66±1.08 and 10.48±2.16,respectively.

Cell lysates from day 5 cultures with and without 1 μM DEX treatmentwere analyzed by Western blotting to test the whether GILZ isoverexpressed in DEX treated cultures. The blot was also reprobed withthe beta-actin antibody as a control to assess equivalent gel loading.As shown in FIG. 13B, the expression of GILZ protein significantlyincreased in the DEX treated samples compared with untreated samples. Inaddition, GILZ fold change caused by DEX is slightly higher in day 8samples than day 5 samples, which is in agreement with the qRT-PCRresults.

Comparison of Dexamethasone with Other Two Glucocorticoids,Hydrocortisone and Prednisolone

Experiments in shake flasks then tested whether the protective effect ofDEX on CHO cell viability was limited to DEX or could be extended toother glucocorticoid compounds, such as hydrocortisone (HYC) andprednisolone (PRD). DEX, HYC and PRD (Sigma-Aldrich, St. Louis, Mo.)were added two days after inoculation at final concentrations of 0, 0.1,1 and 10 μM for each of these three compounds. Peak VCD, final VCD andfinal viability of cultured cells are presented in Table 2.

TABLE 2 Treatment Peak VCD Final VCD Concentration (×10⁶ (×10⁶ Viability(μM) cells/mL) cells/mL) (%) Dexamethasone 0.1 7.99 7.64 72.3 (DEX) 19.02 8.40 75.8 10 7.49 7.38 82.3 Hydrocortisone 0.1 10.06 7.86 60.1(HYC) 1 9.51 8.15 65.1 10 8.44 7.80 75.5 Prednisolone 0.1 8.99 8.26 69.4(PRD) 1 9.25 8.79 76.8 10 8.05 8.27 77.5 Control N/A 8.69 7.56 56.6

Similar to DEX, HYC (60.1-75.5% final viability) and PRD (69.4-75.5%final viability) also showed dose-dependent cell protective effects.Viability on day 10 was 56.6% for the control compared to day 10viabilities with 0.1 and 10 μM concentrations for DEX of 72.3% and82.3%; for HYC of 60.1% and 75.5%; and PRD of 69.4% and 77.5%,respectively. Thus all three glucocorticoid compounds improved cultureviability with maximum effects at 10 μM for all three glucocorticoidcompounds.

The Death-Suppression Action of Dexamethasone Involves GILZ andGlucocorticoid Receptor

In order to determine whether the viability improvement from DEXaddition was mediated through GILZ and the glucocorticoid receptor (GR),the GR antagonist mifepristone (RU-486) was added to cell culture mediumbefore DEX treatment. For conditions with and without RU-486, thepercent increase of final cell viability induced by DEX is shown in FIG.14A. The ability of DEX to improve cell viability was substantiallyreduced in the presence of 1 μM of RU-486. The percent increase of cellviability induced by 0.1 and 1 μM DEX decreased from 30.5% and 32.6%(without RU-486) to 4.7% and 5.7% (with 1 μM RU-486), respectively. Inthe mean time, qRT-PCR analysis (FIG. 14B) shows that in the presence of1 μM RU-486, the upregulation effect of DEX on GILZ expression wassignificantly attenuated. The fold change induced by 0.1 and 1 μM DEXdecreased from 9.0±1.9 and 11.3±3.0 (without RU-486) to 1.8±0.9 and2.3±1.0 (with 1 μM RU-486), respectively. Western Blotting analysis(FIG. 14C) further confirmed the overexpression of GILZ protein wassignificantly decreased in the presence of 1 μM RU-486. These resultsindicate that the mechanism through which DEX increased viabilityinvolved GILZ and was GR-dependent, since RU-486 competes with DEX forthe ligand-binding domain of the GR

Application of Dexamethasone in 10-L Fed Batch Bioreactor Culture

Fed-batch cultures in 10-L bioreactors were carried out to determinewhether the effect of DEX seen in shake flasks would scale tobioreactors. The overall goal of DEX was to suppress cell death, extendculture longevity and consequently increase glycoprotein production fora process in bioreactors. In this study, three bioreactors were operatedusing the same conditions described above; with 1 μM of DEX added to twobioreactors on day 2 or day 7 respectively. Different from shake flaskruns, bioreactor runs were extended to 14 days, the culture temperaturewas shifted to a lower temperature during the late exponential phase ofthe culture, and a proprietary feeding medium was used instead offeeding only glucose and glutamine.

Bioreactors with DEX added on either day 2 or day 7 reached maximum celldensities of approximately 8.3×10⁶ cell/mL on day 8, compared to 7.8×10⁶cell/mL without DEX (FIG. 15A). DEX addition decreased the cell deathrate in the bioreactors. Cell viability was 94% viability for allconditions on day 6 (FIG. 15B). By day 14, the percent viabilitydecreased to 29% without DEX, compared to 55% and 39% with DEX added ondays 2 or 7, respectively. The final VCD on day 14 with DEX added ondays 2 or 7 were 4.1 and 3.5×10⁶ cell/mL respectively, compared to2.8×10⁶ cell/mL without DEX addition. Normalized protein titers wereapproximately 5.5 on day 7 prior to the maximum VCD (FIG. 15C).Thereafter, protein production during the stationary and death phases ofthe cultures was higher in the bioreactors with DEX addition. The day 14harvest normalized titers were 12.5 in both bioreactors with DEX,compared to 10.5 without DEX addition, a 20% increase.

Conclusion

Our effort on medium optimization has led us to unexpectedly discoverthat glucocorticoids can significantly attenuate the cell viabilitydecline in the fed-batch culture of CHO cells. In the mechanism study ofthis phenomenon, the involvement of upregulation of anti-apoptotic geneGILZ was identified through qRT-PCR and Western blot analysis. Bystudying effects of DEX's analogs and antagonist on CHO cell growth, therole of GILZ and glucocorticoid receptor in mediating the action of DEXwas determined. Fed-batch bioreactor experiments demonstrates thisglucocorticoid analog to be an effective, feasible, and cost-efficientchemical for attenuating the viability decline in cell cultures.

Example 4

In this study, the effects of dexamethasone (DEX) on CHO cell growth,protein sialylation and aggregation were studied on another CHO cellline with different glycoprotein (CTLA4lg) secretion.

Cell Line and Medium

The CHO cell line used in this study was originally subcloned from DG44parental cells and cultured in a proprietary chemical-defined growthmedium.

Shake Flask Experiments

The experiments were carried out in 250-mL shake flasks (VWRinternational) with starting volumes of 100 mL and initial celldensities of 6×10⁵ cells/mL. The cultures were placed on a shakerplatform (VWR international) at 150 rpm and maintained at 37° C. and 6%CO₂ for ten days. The cultures were sampled daily and the pH wasadjusted as needed using 1M sodium carbonate and fed with glucose andglutamine every two days in order to maintain them at adequate levels.The glucocorticoids dexamethasone (DEX) was added at finalconcentrations between 0.001-10

M on day 2. Cell density and viability were measured offline using aCedex automated cell counter (Innovatis AG, Bielefeld, Germany). CulturepH and concentrations for glucose and glutamine were measured off-lineusing a Bioprofile Analyzer 400 (Nova Biomedical Corporation, Waltham,Mass.). Supernatants from culture harvests were collected for analysisof sialic acid content and HMW level.

Sialic Acid and HMW Content Assay

The sialic acid and HMW content assay were performed as described in theprevious examples.

Results Effect of Dexamethasone on CHO Cell Growth

The glucocorticoid dexamethasone (DEX) was added to shake flask culturesat final concentrations between 0.001 to 10 μM two days afterinoculation to assess the effects of DEX on CHO cell viability and thepotential to extend the cell culture period. The viable cell density(VCD) profile in the dose-response study (FIG. 16A) shows increasinginhibition of cell growth occurring after DEX treatment, though theconcentration dependency is not obvious in the studied range. The peakVCD reached 13.9×10⁶ cells/mL in the untreated control culture, whereasthe peak VCD ranged from 10.6×10⁶ to 12.7×10⁶ cells/mL in the culturestreated with 0.001 to 10 μM DEX. Cell viability for the untreatedcultures decreased rapidly after Day 6 (FIG. 16B) and percent viabilityon day 10 was only 64.5%. In contrast, the final cell viabilities with0.001 μM, and 10 μM DEX were 75.5% and 82.3%, respectively.

Dexamethasone (DEX) Increases Sialylation and Reduces HMW Level ofGlycoprotein CTLA4lg

Besides the cell growth, the effects of DEX on sialic acid content andHMW level were also assessed. Through comparing with untreated cultures,the percent increase of sialic acid content (FIG. 17A) and the percentreduction of HMW species (FIG. 17B) induced by various concentration ofDEX are shown in FIG. 17. It is obviously that DEX is capable ofincreasing the sialylation and reducing the HMW species of glycoprotein,indicating improved protein quality, even at 0.001 μM concentration. Theconcentration-dependencies of DEX on sialic acid content and HMW speciesare not obvious as the concentration of DEX is above 0.01 μM.

These results demonstrated that the actions of DEX on cell viabilityimprovement, glycoprotein sialylation improvement and aggregationreduction are not limited to either single cell dine or mediumformulation.

Example 5

In this study, the feasibility of utilizing DEX in the cell culturemedium for large scale recombinant glycoprotein production isdemonstrated in 500-L and 5000-L scale bioreactors.

Cell Line and Medium

The CHO cell line used in this study was subcloned from DG44 parentalcells and cultured in a proprietary growth medium.

Bioreactor Operation

Bioreactor experiments were performed in 7-L, 500-L and 5000-Lbioreactors with starting working volumes around 3 L, 300 L and 3000 L,respectively. All the bioreactor runs started at 37° C. and shifted tolower temperature when cells entered production phase in order to extendthe cell culture period. pH, and dissolved oxygen were maintained at7.05, and 50% air saturation. Agitation rates for 7-L, 500-L and 5000-Lscales are 180, 75 and 60 rpm respectively. All the bioreactorexperiments were conducted in a fed batch mode with daily feeding of aprotein-free medium in order to maintain the glucose and other nutrientsat certain levels. Dexamethasone was included in the feed medium at allthe production scales for the purpose of increasing cell viability andprotein sialylation. Samples were taken during the cell culture processand analyzed for cell density, cell viability, substrates andmetabolites.

The titer, sialic acid and HMW content assay were performed as describedin the previous examples.

Results

FIGS. 18A and 18B present the bioreactor performance with respect tocell growth and viability. The cell growth through scale up from 7-L to5000-L was observed to have similar peak viable cell densities between12 to 13×10⁶ cells/mL with error bars representing the standarddeviation of the runs at a particular scale overlapping at every timepoint. Cell densities peaked on Day 7 at 5000-L scale and at Day 8 for7-L and 500-L scales. The Day 14 average viabilities of the cultureswere 88%, 84%, and 91% at 7-L, 500-L, and 5000-L bioreactor scale,respectively. FIGS. 18C and 18D present the productivity and sialic acidprofiles from 7-L, 500-L and 5000-L bioreactor scales. The Day 14 titers(reported as normalized value) were 13.2, 11.6, and 13.6 at 7, 500, and5000-L scales, respectively. Peak sialic acid levels (reported asnormalized value) were 16.0, 18.0 and 19.0 at increasing scales. Thesialic acid dropped by approximately 2.6 units by the end of the runsfor all scales.

Conclusion

Overall, with dexamethasone included in the feed medium performed wellat all scales, indicating the feasibility of utilizing dexamethasone asmedium additives for industrial scale production.

What is claimed is:
 1. A method of increasing sialylation of arecombinant CTLA4 molecule, comprising: a) culturing CHO cells whichproduce a recombinant CTLA4 molecule in cell culture under conditionsthat allow for protein production; and b) feeding the CHO cells withfeeding medium containing dexamethasone, wherein the recombinant CTLA4molecule comprises the extracellular domain of CTLA4 with the amino acidsequence beginning with methionine at position 27 or alanine at position26 and ending at aspartic acid at position 150 as shown in SEQ ID NO: 2joined to an immunoglobulin moiety comprising hinge, CH2 and CH3 domainsand wherein the dexamethasone is sustained or maintained in the CHO cellculture at a concentration of 0.001 μM to 10 μM, wherein the CHO cellculture volume is at least 500 liters, and wherein sialylation of therecombinant CTLA4 molecule is increased compared to sialylationrecombinant CTLA4 molecule in a culture without dexamethasone addition.2. A method of reducing cell death rate in a CHO cell culture producingrecombinant CTLA4 molecule, comprising: a) culturing CHO cells whichproduce a recombinant CTLA4 molecule in cell culture under conditionsthat allow for protein production; and b) feeding the CHO cells withfeeding medium containing dexamethasone, wherein the recombinant CTLA4molecule comprises the extracellular domain of CTLA4 with the amino acidsequence beginning with methionine at position 27 or alanine at position26 and ending at aspartic acid at position 150 as shown in SEQ ID NO: 2joined to an immunoglobulin moiety comprising hinge, CH2 and CH3 domainsand wherein the dexamethasone is sustained or maintained in the CHO cellculture at a concentration of 0.001 μM to 10 μM, wherein the CHO cellculture volume is at least 500 liters, and wherein the cell death rateis reduced compared to the rate of cell death in a culture withoutdexamethasone addition.
 3. A method of increasing cell viability in aCHO cell culture producing recombinant CTLA4 molecule, comprising: a)culturing CHO cells which produce a recombinant CTLA4 molecule in cellculture under conditions that allow for protein production; and b)feeding the CHO cells with feeding medium containing dexamethasone,wherein the recombinant CTLA4 molecule comprises the extracellulardomain of CTLA4 with the amino acid sequence beginning with methionineat position 27 or alanine at position 26 and ending at aspartic acid atposition 150 as shown in SEQ ID NO: 2 joined to an immunoglobulin moietycomprising hinge, CH2 and CH3 domains and wherein the dexamethasone issustained or maintained in the CHO cell culture at a concentration of0.001 μM to 10 μM, wherein the CHO cell culture volume is at least 500liters, and wherein cell viability is increased compared to cellviability in a culture without dexamethasone addition.
 4. A method ofincreasing titer of a recombinant CTLA4 molecule produced in a CHO cellculture, comprising: a) culturing CHO cells which produce a recombinantCTLA4 molecule in cell culture under conditions that allow for proteinproduction; and b) feeding the CHO cells with feeding medium containingdexamethasone, wherein the recombinant CTLA4 molecule comprises theextracellular domain of CTLA4 with the amino acid sequence beginningwith methionine at position 27 or alanine at position 26 and ending ataspartic acid at position 150 as shown in SEQ ID NO: 2 joined to animmunoglobulin moiety comprising hinge, CH2 and CH3 domains and whereinthe dexamethasone is sustained or maintained in the CHO cell culture ata concentration of 0.001 μM to 10 μM, wherein the CHO cell culturevolume is at least 500 liters, and wherein recombinant CTLA4 moleculetiter is increased compared to recombinant CTLA4 molecule titer in aculture without dexamethasone addition.
 5. A method of decreasingrecombinant CTLA4 molecule aggregation in a CHO cell culture,comprising: a) culturing CHO cells which produce a recombinant CTLA4molecule in cell culture under conditions that allow for proteinproduction; and b) feeding the CHO cells with feeding medium containingdexamethasone, wherein the recombinant CTLA4 molecule comprises theextracellular domain of CTLA4 with the amino acid sequence beginningwith methionine at position 27 or alanine at position 26 and ending ataspartic acid at position 150 as shown in SEQ ID NO: 2 joined to animmunoglobulin moiety comprising hinge, CH2 and CH3 domains and whereinthe dexamethasone is sustained or maintained in the CHO cell culture ata concentration of 0.001 μM to 10 μM, wherein the CHO cell culturevolume is at least 500 liters, and wherein recombinant CTLA4 moleculeaggregation is decreased compared to recombinant CTLA4 moleculeaggregation in a culture without dexamethasone addition.
 6. The methodof any one of claims 1 to 5 wherein the dexamethasone is sustained ormaintained in the CHO cell culture at a concentration of 0.01 μM to 10μM.
 7. The method of any one of claims 1 to 5 wherein the dexamethasoneis sustained or maintained in the CHO cell culture at a concentration of0.1 μM to 10 μM.
 8. The method of any one of claims 1 to 5 wherein thesoluble CTLA4 molecule is CTLA4lg comprising an amino acid sequencebeginning with methionine at position 27 or alanine at position 26 andending at lysine at position 383 as shown in SEQ ID NO:
 2. 9. A cellculture process for the production of a recombinant CTLA4 molecule,comprising: a) culturing CHO cells which produce a recombinant CTLA4molecule in cell culture under conditions that allow for proteinproduction; and b) feeding the CHO cells with feeding medium containingdexamethasone, wherein the recombinant CTLA4 molecule an amino acidsequence beginning with methionine at position 27 or alanine at position26 and ending at lysine at position 383 as shown in SEQ ID NO: 2 andwherein the dexamethasone is sustained or maintained in the CHO cellculture at a concentration of 0.001 μM to 10 μM, and wherein the CHOcell culture volume is at least 500 liters.
 10. The cell culture processof claim 9 wherein the dexamethasone is sustained or maintained in theCHO cell culture at a concentration of 0.01 μM to 10 μM.
 11. The cellculture process of claim 9 wherein the dexamethasone is sustained ormaintained in the CHO cell culture at a concentration of 0.1 μM to 10μM.